KR102081699B1 - 스쿠알렌 생성능을 가지는 형질전환된 코리네박테리움 글루타미쿰 - Google Patents

스쿠알렌 생성능을 가지는 형질전환된 코리네박테리움 글루타미쿰 Download PDF

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KR102081699B1
KR102081699B1 KR1020180005193A KR20180005193A KR102081699B1 KR 102081699 B1 KR102081699 B1 KR 102081699B1 KR 1020180005193 A KR1020180005193 A KR 1020180005193A KR 20180005193 A KR20180005193 A KR 20180005193A KR 102081699 B1 KR102081699 B1 KR 102081699B1
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squalene
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corynebacterium glutamicum
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우한민
박재현
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성균관대학교산학협력단
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Abstract

본 발명은 스쿠알렌 생성능을 가지는 형질 전환된 코리네박테리움 글루타미쿰 균주 이의 용도에 관한 것으로, 상기 균주의 대사경로에 관여하는 유전자를 재조합하여 형질전환 벡터를 제조하고, 이를 이용하여 형질전환한 코리네박테리움 균주에 대한 것이다. 상기 균주를 이용하면, 순도가 높은 상태의 스쿠알렌을 대량으로, 안정적으로 생산할 수 있어, 경제적인 효과를 가진다.

Description

스쿠알렌 생성능을 가지는 형질전환된 코리네박테리움 글루타미쿰 {Transformed corynebacterium glutamicum having capability of producing squalene}
본 발명은 스쿠알렌 생성능을 가지는 형질 전환된 코리네박테리움 글루타미쿰 균주 이의 용도에 관한 것으로, 상기 균주의 대사경로에 관여하는 유전자를 재조합하여 형질전환 벡터를 제조하고, 이를 이용하여 형질전환한 코리네박테리움 균주에 대한 것이다.
스쿠알렌은 탄소 30개 및 수소 50 개가 이중 결합으로 연결된 트리테르페노이드계 불포화 탄화수소화합물로 자연계에서 심해 상어간유에서 발견되며 식물유에 미량 함유된 물질이다. 본 물질은 항산화 기능, 항암 효과, 면역 증진 기능 등의 건강기능성을 가지고 있어 건강기능식품, 화장품, 의약품 등에 사용 되는 유용 물질이다. 스쿠알렌을 생산하기 위하여 심해 상어를 무분별하게 포획하는 것이 금지 되면서 이를 대체하기 위해 식물로부터 스쿠알렌을 추출이 진행되었다. 하지만 식물에는 스쿠알렌이 미량 함유되어있어 식물로부터 추출되는 스쿠알렌 생산은 비용적인 면에서 효과적이지 못하며, 공기 중금속에 의해서 식물배양시 오염의 가능성이 있다고 알려져 있다. 최근 뷰티케어 산업이 급격하게 성장하여 향후 스쿠알렌 소비가 증가될 것이 예상되고 있어, 경제적이고 안정적인 방법으로 지속 가능한 스쿠알렌의 대량 생산 대체 방법이 필요하다.
코리네박테리움 글루타미쿰은 유전자 조작을 통하여 대사경로 및 대사경로를 조절하는 것이 가능하며, 이를 이용하여 합성생물학/대사공학적 기법을 통해 기존의 대사경로를 조절하거나 새로운 대사경로를 이용하여 아미노산등 대사 산물을 대량 생산하는 산업 균주로 알려져 있다. 따라서, 코리네박테리움 글루타미쿰의 대사 경로에 관여하는 유전자를 재조합 및 편집하여 원하는 대사 산물을 생산할 수 있고, 특히 상기 균주를 이용하여 스쿠알렌을 대량으로 생산할 수 있는 방법에 대한 연구가 필요한 실정이다.
한국출원 제10-2017-0099545호 한국출원 제10-2016-0041774호
Choi SY, Lee HJ, Choi J, Kim J, Sim SJ, Um Y, Kim Y, Lee TS, Keasling J, Woo HM (2016) Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria. Biotechnol Biofuels 69:202 Kang, M. K., Lee, J., Um, Y., Lee, T. S., Bott, M., Park, S. J., & Woo, H. M. (2014). Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization. Applied microbiology and biotechnology, 98(13), 5991-6002.)
이에 본 발명자들은 대사 산물로서 스쿠알렌을 생성하는 능력을 가지도록 형질전환된 코리네박테리움 글루타미쿰에 대한 본 발명을 완성하였다.
따라서, 본 발명의 목적은 스쿠알렌 생산에 관여하는 유전자를 포함하는 벡터로 형질 전환 되며, 본래의 대사 과정에 관여하는 일부 유전자의 발현을 저해하여 스쿠알렌 생성능을 향상시킨, 코리네박테리움 글루타미쿰 균주를 제공하는 것이다.
또한, 본 발명의 다른 목적은 상기 형질전환된 코리네박테리움 글루타미쿰을 배양하여 스쿠알렌을 생산하는 방법을 제공하는 것이다.
본 발명의 또 다른 목적은 상기 형질전환된 코리네박테리움 글루타미쿰의 스쿠알렌 생성능을 향상시키기 위한 배양액 조성물을 제공하는 것이다.
상기와 같은 과제를 해결하기 위하여, 본 발명은 피루베이트(pyruvate) 및 D-글리세르알데하이드 3-포스페이트(D-glyceraldehyde 3-phosphate, G3P)로부터 1-데옥시-D-크실룰로오스 5-포스페이트(1-deoxy-D-xylulose 5-phosphate, DXP)를 생산하는 효소를 코딩하는 유전자; 아이소펜테닐 디포스페이트(isophentenyl diphosphate, IPP)로부터 디메틸알릴 디포스페이트(dimethylallyl diphosphate, DMAPP)를 생산하는 효소를 코딩하는 유전자; 디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(farnesyl diphosphate, FPP)를 생산하는 효소를 코딩하는 유전자; 및 파네실 디포스페이트(FPP)로부터 스쿠알렌(squalene)을 생산하는 효소를 코딩하는 유전자를 포함하는 코리네박테리움 글루타미쿰 균주를 제공한다.
본 발명의 일 실시예에서, 피루베이트(pyruvate) 및 D-글리세르알데하이드 3-포스페이트(D-glyceraldehyde 3-phosphate, G3P)로부터 1-데옥시-D-크실룰로오스 5-포스페이트(1-deoxy-D-xylulose 5-phosphate, DXP)를 생산하는 효소를 코딩하는 유전자는 서열번호 1의 염기서열을 포함하고, 아이소펜테닐 디포스페이트(isophentenyl diphosphate, IPP)로부터 디메틸알릴 디포스페이트(dimethylallyl diphosphate, DMAPP)를 생산하는 효소를 코딩하는 유전자는 서열번호 2의 염기서열을 포함하고, 디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(farnesyl diphosphate, FPP)를 생산하는 효소를 코딩하는 유전자는 서열번호 3의 염기서열을 포함하며, 파네실 디포스페이트(FPP)로부터 스쿠알렌(squalene)을 생산하는 효소를 코딩하는 유전자는 서열번호 4의 염기서열을 포함한다.
또한, 본 발명의 일 실시예에서, 상기 피루베이트 및 D-글리세르알데하이드 3-포스페이트로(G3P)부터 1-데옥시-D-크실룰로오스 5-포스페이트(DXP)를 생산하는 효소는, 데옥시크실룰로오스-5-포스페이트 합성효소 (deoxyxylulose-5-phosphate synthase, dxs)이고, 아이소펜테닐 디포스페이트로(IPP)부터 디메틸알릴 디포스페이트(DMAPP)를 생산하는 효소는, 아이소펜테닐-디포스페이트 델타 아이소머라아제(isopentenyl diphosphate delta isomerase, idi)이고, 디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(FPP)를 생산하는 효소는, 제라닐트랜스퍼라아제(geranyl transferase, ispA)이며, 파네실 디포스페이트(FPP)로부터 스쿠알렌을 생산하는 효소는, 스쿠알렌 합성 효소이다.
본 발명의 다른 실시예에서 본 발명의 코리네박테리움 글루타미쿰은 상기 효소를 각각 코딩하는 유전자인 dxs 유전자, idi 유전자, ispA 유전자 및, 스쿠알렌 합성효소를 코딩하는 유전자로부터 유래된 서열인 trSQS(CgCO)를 포함하는 벡터로 형질 전환된 것이다. (이를 제 1 벡터라 한다.)
본 발명의 또 다른 실시예에서, 상기 dxs 유전자 및 idi 유전자는 코리네박테리움 글루타미쿰으로부터 유래된 것이며, ispA 유전자는 대장균(E.coli)로부터 유래된 것이고, 스쿠알렌 합성효소 유전자는 사카로마이세스 세레비지에(Saccharomyces cerevisiae)로부터 유래된 것이나, 이에 제한되는 것은 아니다.
본 발명의 코리네박테리움 글루타미쿰은 상기 언급된 효소들을 코딩하는 유전자를 포함하며, 이에 추가적으로 디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자(‘idsA 유전자’), D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자(‘gapA 유전자’) 또는 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자(‘gdh 유전자’)의 발현을 저해하는 sgRNA를 함유한 벡터를 함께 포함한다. (이를 제 2 벡터라 한다.)
본 발명의 일 실시예에서, 상기 디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자는 서열번호 6 또는 서열번호 7의 염기서열을 포함하고, D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자는 서열번호 8 또는 서열번호 9의 염기서열을 포함하고, 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자는 서열번호 10 또는 서열번호 11의 염기서열을 포함하나, 이에 제한되는 것은 아니다.
본 발명의 다른 실시예에서 상기 디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자, D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자 또는 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자의 발현을 저해시키기 위하여, 크리스퍼 유전자 조절 시스템을 사용하였으며, 이를 위하여 상기　제 2 벡터는 dCas9 단백질을 코딩하는 유전자를 포함한다.
즉, 본 발명의 코리네박테리움 글루타미쿰 균주는 제1벡터 및 제2 벡터로 형질전환된 것으로서, 상기 dxs 유전자, idi 유전자, ispA 유전자 및 스쿠알렌 합성효소 유래 서열인 trSQS(CgCO)를 포함하고, 상기 제2벡터는 디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자, D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자 또는 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자의 발현을 저해하는 sgRNA 및 dCas9 유전자를 포함한다.
위와 같은 벡터로 형질전환된 본 발명의 코리네 박테리움 글루타미쿰은 기탁번호 KCCM12189P인 균주이다.
다른 양태로서 본 발명은 상기 균주를 배양하는 단계를 포함하는 스쿠알렌 생산 방법에 대한 것이다.
본 발명의 일 실시예에서, 상기 균주는 질소 성분을 낮춘 배양액으로부터 배양되며, 스쿠알렌의 생산성을 높이기 위하여, 배양액에 트립톤을 포함할 수 있다.
본 발명의 코리네박테리움 글루타미쿰은 대사 경로를 조절하는 유전자를 재조합하여 스쿠알렌을 생성능을 가지도록 형질전환된 것이며, 특히 스쿠알렌 합성에 관여하는 효소를 코딩하는 유전자를 도입하고, 반대로 스쿠알렌 합성을 저해할 수 있는 효소를 코딩하는 유전자는 발현을 저해하여, 스쿠알렌 생산능을 더욱 향상시킨 특징을 가진다. 본 발명의 균주는 순도가 높은 스쿠알렌을 안정적으로 생산할 수 있으며, 대량으로 생산하는데 적합하다.
특히, 상기 유전자 발현 저해를 위하여, 크리스퍼 유전자 조절 시스템을 도입함으로써, 보다 손쉽게 스쿠알렌 생성능이 향상된 형질전환 균주를 만들어낼 수 있고, 나아가 상기 형질전환 균주로부터 스쿠알렌을 생성하기에 적합한 배양액을 제공할 수 있다.
도 1은 형질 전환된 코리네박테리움 글루타미쿰 균주의 스쿠알렌 생산경로를 도식화한 것이다. 각 과정에 관여하는 유전자의 이름 및 유래 균주에 대해 기재되어 있으며, 이들의 발현을 조절하여 스쿠알렌을 생산할 수 있다.
도 2는 제1벡터(왼쪽) 및 제2벡터(오른쪽)을 도식화한 것이다.
도3은 형질전환 균주가 생산한 물질이 스쿠알렌임을 확인한 불꽃 이온화 검출기 가스크로마토그래피 결과를 나타낸 것이다. 위쪽에 도시된 것은 스쿠알렌 스탠다드(Sigma Aldrich, St. Louis, Missouri, United States)를 확인한 것이고, 아래쪽에 도시된 것은 본원 발명의 제1벡터 및 제2벡터를 모두 포함하여 형질전환된 균주로부터 생산되는 스쿠알렌을 확인한 결과이다.
도4는 스쿠알렌 합성효소 유전자(trSQS 유전자), 데옥시자일룰로오스-5-포스페이트 합성효소 유전자(dxs 유전자) 디메틸알릴 디포스페이트 합성효소 유전자(idi 유전자) 파네실 디포스페이트 합성 효소(ispA 유전자)를 포함하는 제 1벡터와 sgRNA를 포함하지 않는 제 2벡터로 형질전환 된 균주의 스쿠알렌 생산량을 확인한 결과이다.
도 5는 스쿠알렌 합성 효소 발현 유전자(trSQS 유전자), 데옥시자일룰로오스-5-포스페이트 합성 효소 발현 유전자(dxs 유전자) 디메틸알릴 디포스페이트 합성 효소 발현 유전자 (idi 유전자) 파네실 디포스페이트 합성 효소 발현 유전자 (ispA 유전자)를 포함하는 제 1벡터와 크리스퍼 유전자 발현 저해 기술을 적용한 벡터를 포함하는 제 2벡터로 형질 전환 된 균주의 스쿠알렌 생산량을 확인한 것이다.
도 6은 스쿠알렌 합성 효소 발현 유전자(trSQS 유전자), 데옥시자일룰로오스-5-포스페이트 합성 효소 발현 유전자(dxs 유전자) 디메틸알릴 디포스페이트 합성 효소 발현 유전자 (idi 유전자) 파네실 디포스페이트 합성 효소 발현 유전자 (ispA 유전자)를 갖는 제 1벡터를 포함하는 균주(Control)와 제 1벡터와 함께 크리스퍼 유전자 발현 저해 벡터인 제 2벡터로 형질 전환 된 균주의 CGXII 최소배지, CGXII 최소배지의 질소성분을 낮춘 배지(N), CGXII 최소배지에 tryptone을 첨가한 배지, 2YT 배지에서 배양한 균주의 스쿠알렌 생산량을 확인한 결과를 나타낸 것이다.
도 7은 본 발명의 제1벡터의 개열지도를 도식화하여 나타낸 것이다.
도 8은 본 발명의 제2벡터 중, sgRNA-idsA-r1(a)　및 sgRNA-idsA-r3(b)가 삽입된 벡터의 개열지도를 도식화하여 나타낸 것이다.
도 9는 본 발명의 제2벡터 중, sgRNA-gapA-r1(a) 및 sgRNA-gapA-r2(b)가 삽입된 벡터의 개열지도를 도식화하여 나타낸 것이다.
도 10은 본 발명의 제2벡터 중, sgRNA-gdh-r1(a) 및 sgRNA-gdh-r2(b)가 삽입된 벡터의 개열지도를 도식화하여 나타낸 것이다.
본 발명자들은 스쿠알렌 생성능이 향상된 신규 균주를 개발하기 위하여,
피루베이트(pyruvate) 및 D-글리세르알데하이드 3-포스페이트(D-glyceraldehyde 3-phosphate, G3P)로부터 1-데옥시-D-크실룰로오스 5-포스페이트(1-deoxy-D-xylulose 5-phosphate, DXP)를 생산하는 효소를 코딩하는유전자, 아이소펜테닐 디포스페이트(isophentenyl diphosphate, IPP)로부터 디메틸알릴 디포스페이트(dimethylallyl diphosphate, DMAPP)를 생산하는 효소를 코딩하는 유전자, 디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(farnesyl diphosphate, FPP)를 생산하는 효소를 코딩하는 유전자 및 파네실 디포스페이트(FPP)로부터 스쿠알렌(squalene)을 생산하는 효소를 코딩하는 유전자를 포함하는 제1벡터; 및
*디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자, D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자 또는 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자의 발현을 저해하는 sgRNA를 포함하는 벡터를 추가적으로 포함하는 제2벡터로 동시에 코리네박테리움 글루타미쿰 균주를 형질전환 하고, 상기 균주가 스쿠알렌 생성능을 가지는 것을 확인하여, 2017년 12월 19일 한국미생물보존센터에 수탁번호 KCCM12189P로 기탁하였다.
즉, 상기 균주는 이에 제한되는 것은 아니나, 제1 벡터 및/또는 제2 벡터로 형질전환된 것일 수 있다. ‘제1 또는 제2’라는 표현은, 벡터의 종류를 구별하기 위한 것일 뿐, 형질전환의 순서나 방법을 제한하는 것이 아니다
상기 제1벡터는, 피루베이트(pyruvate) 및 D-글리세르알데하이드 3-포스페이트(D-glyceraldehyde 3-phosphate, G3P)로부터 1-데옥시-D-크실룰로오스 5-포스페이트(1-deoxy-D-xylulose 5-phosphate, DXP)를 생산하는 효소인 데옥시크실룰로오스-5-포스페이트 합성효소 (deoxyxylulose-5-phosphate synthase,) 유전자(‘dxs 유전자’), 아이소펜테닐 디포스페이트(isophentenyl diphosphate, IPP)로부터 디메틸알릴 디포스페이트(dimethylallyl diphosphate, DMAPP)를 생산하는 효소인 아이소펜테닐-디포스페이트 델타 아이소머라아제(isopentenyl diphosphate delta isomerase)유전자(‘idi유전자’), 디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(farnesyl diphosphate, FPP)를 생산하는 효소인 제라닐트랜스퍼라아제(geranyl transferase)유전자 (‘ispA유전자’) 및 파네실 디포스페이트(FPP)로부터 스쿠알렌(squalene)을 생산하는 효소를 코딩하는 유전자로부터 유래된 서열인 trSQS(CgCO)를 포함한다. 또한, 이에 제한되는 것은 아니나, 상기 dxs 유전자는 코리네박테리움 글루타미쿰 유래의 서열번호 1의 염기서열을 포함하고, 상기 idi유전자는 코리네박테리움 글루타미쿰 유래의 서열번호 2의 염기서열을 포함하고, 상기 ispA 유전자는 대장균 유래의 서열번호 3의 염기서열을 포함하며, 상기 trSQS(CgCO) 서열은 사카로마이세스 세레비지에로부터 유래된 서열번호 4의 염기 서열을 포함한다.
또한, 상기 제1벡터는 서열번호 5의 서열을 포함할 수 있다.
본 발명의 일 실시예에서, 상기 제1벡터만으로 형질전환한 코리네박테리움 글루타미쿰의 경우, 스쿠알렌 생산능을 가진다. 즉, 형질전환 벡터에, trSQS(CgCO) 서열을 포함하고, dxs, idi, ispA 유전자가 삽입된 제1벡터 및 sgRNA를 포함하지 않는 제2벡터로 형질전환된 코리네박테리움 글루타미쿰을 배양하여 불꽃이온화검출기로 확인한 결과, 도 4 에서 보듯이, 스쿠알렌 피크가 확인되었다. 따라서, 제1벡터 만으로 형질전환된 코리네박테리움 글루타미쿰 역시 스쿠알렌 생산능을 가진다.
상기 제1벡터에 삽입되는 유전자 중, dxs 및 idi 유전자는 코리네박테리움 글루타미쿰으로부터 유래된 것, ispA 유전자는 대장균(E.coli)으로부터 유래된 것, 스쿠알렌 합성효소는 사카로마이세스 세레비지에(Saccharomyces cerevisiae)로부터 유래된 것일 수 있다.
본 발명의 제2벡터는 디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트(GGPP)를 생산하는 효소를 코딩하는 유전자(‘idsA 유전자’), D-글리세르알데히드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트(1,3BPG)를 생산하는 효소를 코딩하는 유전자(‘gapA 유전자’) 또는 알파케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자(‘gdh 유전자’)의 발현을 저해할 수 있는 sgRNA 및 dCas9 유전자를 포함하는 벡터이다.
상기 제2벡터를 통해 발현을 저해하는 유전자로서, idsA 유전자는 서열번호 6 또는 서열번호 7의 염기서열을 포함하고, gapA 유전자는 서열번호 8 또는 서열번호 9의 염기서열을 포함하며, gdh 유전자는 서열번호 10 또는 서열번호 11의 염기서열을 포함할 수 있다.
상기 제2벡터는 상기 효소를 코딩하는 유전자 유래의 프로모터를 각각 포함할 수 있으며, 각각의 유전자의 프로모터 부근에 위치하며, 비 주형 가닥을 타겟팅하는 서열 중 1 이상을 선택하여 포함할 수 있다. 상기 프로모터 서열을 유전자당 2개씩 선정하였으며, 이들을 각각 idsA-r1(서열번호 12), idsA-r3(서열번호 13), gapA-r1(서열번호 14), gapA-r2(서열번호 15), gdh-r1(서열번호 16), gdh-r2(서열번호 17)라고 명명하였다.
또한 제2벡터에 포함되는 dCas9 유전자는 핵산분해효소가 없는 Cas9 단백질을 코딩하는 유전자이며, sgRNA의 서열에 따라 특정 DNA에 결합할 수 있는 능력을 가졌으나, 타겟 DNA를 자르는 능력은 없다. 따라서, 서열을 바꾸지 않으면서 전사과정을 직접 조절하는 역할을 수행한다.
상기 제2벡터는 바람직하게는 서열번호 18 내지 서열번호 23의 서열 중 선택된 것일 수 있으나, 이에 제한되는 것은 아니다.
*본 발명의 일 실시예에서, 상기 제1벡터 및 제2벡터를 모두 포함하여 형질전환된 코리네박테리움 글루타미쿰은, 제1벡터만으로 형질전환된 균주에 비하여 향상된 스쿠알렌 생성능을 가진다. 제2벡터에는 상기 서열번호 6 내지 서열번호 11의 서열 중에서 선택된 서열이 포함되며, 각 유전자 발현 억제 시, 스쿠알렌 생산 효율에는 차이가 있을 수 있다. 그러나, 제1벡터와 제2벡터를 동시에 형질전환 할 경우, 제1벡터만으로 형질 전환한 경우에 비하여 높은 스쿠알렌 생성능을 가지는 것을 확인하였다. (도 5)
본 발명의 명세서에 개시된 모든 벡터 내의 유전자들은 서로 작동 가능하게 연결된다. 작동 가능하다는 것의 의미는 타겟 유전자가 정상적으로 발현될 수 있음을 의미하는 것이다
또한 본 명세서에서 타겟 유전자란, 균주 내에서 발현되어 해당 유전자의 기능이 발휘되기 위해 벡터 내에 삽입된 유전자를 의미할 수 있다 상기 균주는, 상기 제1 벡터 및/또는 제2 벡터가 모균주인 코리네박테리움 글루타미쿰 (기탁번호: ATCC 13032)에 형질전환된 것일 수 있다. 모균주에는, 제1 벡터만 도입될 수 있고, 제1 벡터와 제2 벡터가 모두 도입될 수도 있다.
상기 형질전환된 균주는 기탁번호 KCCM12189P인 균주일 수 있다. 기탁번호 KCCM12189P 균주는 모균주인 코리네박테리움 글루타미쿰을 제1벡터인 pBbEB1c-trSQS(CgCO)-dxs-idi-ispA, 및 제2벡터인 pCoryne-sgRNA target-r으로 형질전환된 균주를 의미하는 것이다.
다른 측면에서, 본 발명은 상기 형질전환된 코리네박테리움 글루타미쿰 균주를 배양하는 단계를 포함하는, 스쿠알렌 생산 방법이다.
상기 배양 방법은 본 기술분야에서 통상적으로 사용하는 방법에 의하여 수행될 수 있으며, 보다 바람직하게는 플라스크(baffled-flask)를 이용한 진탕배양 또는 바이오렉터(bioreactor)를 이용하여 배양할 수 있다. 상기 바이오렉터 이용 시, 배양 온도는 29 내지 31 ℃, 120 내지 900rpm 조건에서 12 내지 60 시간동안 배양할 수 있고, 본 발명의 일 실시예에서는 30℃, 900rpm 조건에서 배양 후, 스쿠알렌 생산량을 측정하였다.
또 다른 측면에서 본 발명은, 상기 형질전환된 코리네박테리움 글루타미쿰 균주를 배양하는 배양액 조성물에 대한 것이다.
상기 배양액은 CgXII 최소배지를 기본으로 하고 있으며, 질소량을 조절하기 위하여 황산암모늄 및 요소의 양을 한정한 배양액 조성물, 트립톤을 배지에 포함하는 배양액 조성물, 또는 이스트 추출물 및 염화나트륨을 추가로 포함하는 배양액 조성물 중 선택할 수 있다.
이에 제한되는 것은 아니나, 상기 황산 암모늄 0.001 내지 0.1 g/L 및 요소 0.01 내지 1.0 g/L를 사용하며, 보다 바람직하게는 황산 암모늄 0.01g/L 및 요소 0.5g/L를 사용한다. 상기 범위는 배양액 내 질소량을 조절하기 위한 것이다. 상기 범위 이하로 포함될 경우, 균주를 배양하기 위한 질소 농도가 지나치게 적은 문제가 있고, 상기 범위 이상으로 포함될 경우에는 질소량 조절에 의한 영향이 없다.
또한, 이에 제한되는 것은 아니나, 상기 트립톤의 경우, 배양액 내 10 내지 20g/L로 포함될 수 있으며, 보다 바람직하게는 15 g/L로 포함될 수 있다.
아울러, 상기 배양액 내에 추가적으로, 이스트 추출물은 1g/L 내지 20g/L, 염화나트륨은 1g/L 내지 10g/L으로 포함될 수 있으며, 보다 바람직하게는 이스트 추출물 10g/L, 염화나트륨 5g/L 로 포함될 수 있다.
상기 배양액의 조성은 본 발명의 형질전환된 코리네박테리움 글루타미쿰의 스쿠알렌 생산량이 최대치일 때의 조성이며, 배양액 내 성분에 따라 스쿠알렌의 생산량이 달라질 수 있다.
이하 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
<실시예 1>
스쿠알렌 생산을 위한 유전자 선별
아이소프레노이드(isoprenoid) 대사 경로를 활용하여 스쿠알렌을 생산하기 위하여, 선행 논문(Choi SY, Lee HJ, Choi J, Kim J, Sim SJ, Um Y, Kim Y, Lee TS, Keasling J, Woo HM (2016) Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria. Biotechnol Biofuels 69:202)에 기재된 내용에 따라, dxs 유전자, idi 유전자, ispA 유전자를 이용하여 스쿠알렌에 이르는 대사경로를 코리네박테리움 글루타미쿰에 적용하였다. 상기 dxs 유전자, idi 유전자는 코리네박테리움 글루타미쿰으로부터 유래하였으며, 상기 ispA 유전자는 대장균(E. coli)로부터 유래한 것이다.
또한, FPP로부터 스쿠알렌을 생산하는 생산하는 효소를 코딩하는 유전자는 사카로마이세스 세레비지에(Saccharomyces cerevisiae)에서 유래된 것으로, ERG9 유전자의 DNA 시퀀스 3’으로부터 72bp 가 제거된 유전자를 코리네박테리움 글루타미쿰에 맞추어 코돈 최적화 과정을 거친 후 Genscriptⓡ에서 스쿠알렌 합성 시퀀스 trSQS(CgCO)를 주문 제작 하였다. (Zhang, D. L., Jennings, S. M., Robinson, G. W., & Poulter, C. D. (1993). Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Archives of biochemistry and biophysics, 304(1), 133-143.)
<실시예 2>
스쿠알렌 합성 유전자 발현 벡터(‘제1벡터’) 및 형질전환 균주 제작
CoryneBrick 벡터인 pBbEb1c-rfp (Kang, M. K., Lee, J., Um, Y., Lee, T. S., Bott, M., Park, S. J., & Woo, H. M. (2014). Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization. Applied microbiology and biotechnology, 98(13), 5991-6002.)의 rfp 부분을 EcoRI-BamHI 제한효소를 이용하여 제거한 후 그 자리에 스쿠알렌 합성 효소 유전자(trSQS 유전자)를 삽입하여 스쿠알렌을 생산하기 위한 벡터 pBbEB1c-trSQS(CgCO)를 제작하였다.
제작된 pBbEB1c-trSQS(CgCO)에 BglII-BamHI-XhoI 제한효소를 이용하여 코리네박테리움 글루타미쿰의 dxs 유전자의 DNA시퀀스가 삽입된 pBbEB1c-trSQS(CgCO)-dxs벡터, 코리네박테리움 글루타미쿰 dxs 유전자, idi 유전자의 DNA시퀀스가 삽입된 pBbEB1c-trSQS(CgCO)-dxs-idi 벡터, 코리네박테리움 글루타미쿰 dxs , idi 유전자와 대장균의 ispA 유전자의 DNA시퀀스가 삽입된 pBbEB1c-trSQS(CgCO)-dxs-idi-ispA 벡터를 도 2a와 같이 제작하였다.
상기 벡터의 형질전환 여부는 PCR (5’→3’프라이머 시퀀스 :forward-GAG CTC GAT ATC AAA TTA CG /reverse-GAG CTA AGG AAG CTA AAA TG)을 통해 확인하였다.
<실시예 3>
스쿠알렌 생산 균주 내 유전자 발현 억제를 위한 벡터(‘제2벡터’) 및 유전 자 저해 시스템 설계
디메틸알릴 디포스페이트(DMAPP)로부터 제라닐제라닐 파이로포스페이트 (GGPP)를 생산하는 효소를 발현하는 유전자, D-글리세르알데하이드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트 (1,3BPG)를 생산하는 효소를 발현하는 유전자, 알파 케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 발현하는 유전자를 저해하는 sgRNA를 만들기 위하여 각 유전자의 프로모터 부근에 위치하며 비주형가닥을 타겟팅하는 20bp의 protospace를 유전자당 2가지 씩 선정하였다. (5’→3’idsA-r1(서열번호 12): CTC ATC ACA CTA AGA TAC CC, idsA-r3: AGA CGT CCT TCA ATC GAA TA, gdh-r1: TAA ATG CTC ACG TTC AAT TA, gdh-r2: GAG GGC ACC ATG TAC ATC CT, gapA-r1: CAA CCT GTC CTC AAC TTT AG, gapA-r2: CGC ACC AAA CCT ACT CAC AT)
해당 서열을 포함하는 프라이머를 제작하여 pgRNA-bacteria (Addgene Plasmid #44251)플라스미드를 주형으로 PCR을 진행하여 pgRNA-target-r을 제작하였다. 제작된 sgRNA unit을 EcoRI, BamHI 제한효소를 이용하여 유전자 발현 조절 벡터인 pCoryne-sgRNA-dCas9 벡터에 삽입하여 제 2벡터를 제작하였다.
디메틸알릴 디포스페이트(DMAPP) 로부터 제라닐제라닐 파이로포스페이트 (GGPP)를 생산하는 효소를 코딩하는 유전자의 발현을 저해하기 위한 sgRNA를 포함하는 pCoryne-sgRNA-idsA-r1-dCas9, pCoryne-sgRNA-idsA-r3-dCas9 벡터, D-글리세르알데하이드 3-포스페이트(G3P)로부터 1,3-비스포스포글리세레이트 (1,3BPG)를 생산하는 효소를 코딩하는 유전자의 발현을 저해하기 위한 sgRNA를 포함하는 pCoryne-sgRNA-gapA-r1-dCas9, pCoryne-sgRNA-gapA-r2-dCas9 벡터, 알파 케토글루타레이트(α-KG)로부터 글루타메이트를 생산하는 효소를 코딩하는 유전자의 발현을 저해하기 위한 sgRNA를 포함하는 pCoryne-sgRNA-gdh-r1-dCas9, pCoryne-sgRNA -gdh-r2-dCas9 벡터를 제작하였다. 형질전환 여부는 PCR(5’→3’프라이머 시퀀스 : forward-CCC GAA CAA AAA CTC ATC TC/ reverse-GCC GCC ATT ATT ACG ACA AG)을 통해 확인하였다.
<실시예 4>
제1벡터에 의해 형질전환된 코리네박테리움 글루타미쿰 균주의 스쿠알렌 생 산능
형질 전환된 균주들을 일정 시간 배양하여 스쿠알렌의 생산여부를 확인하였다. 플라스크 배양의 배양조건은 250 mL baffled flask에 2% 글루코오스를 포함하는 CgXII 최소배지 50 mL에 균주를 처음 배양 시 OD 600을 1로 맞추어 접종해주었고, 30 ℃의 인큐베이터에서 120 rpm으로 진탕 배양하였다. 배양 시 카나마이신 항생제 25 μg/mL 와 클로람페니콜 항생제 7.5 μg/mL를 넣어주었으며, 벡터의 유전자 발현을 위한 인듀서 1 mM IPTG, 100 nM aTc 를 첨가하였다.
형질전환 균주가 스쿠알렌을 생산하는지 여부를 알아보기 위해 배양된 균주를 유리구슬을 이용하여 세포를 파쇄한 뒤 추출 용매인 클로로포름: 메탄올(2:1)을 이용하여 스쿠알렌을 추출하였다. 추출된 스쿠알렌은 내부표준물질 20 μg/mL 의 1-페닐 옥타디케인을 첨가 후 GC-FID분석하였다.
trSQS 유전자가 삽입된 제 1벡터 또는 trSQS 유전자와 dxs 유전자가 삽입된 제 1벡터 또는 trSQS 유전자와 dxs 유전자, idi 유전자가 삽입된 제 1벡터와 함께 sgRNA를 포함하지 않는 제 2벡터를 가지는 형질 전환된 균주에서는 스쿠알렌의 피크가 확인되지 않았다. 하지만 trSQS 유전자와 dxs 유전자, idi 유전자, ispA 유전자가 삽입된 제 1벡터와 sgRNA를 포함하지 않는 제 2벡터를 가지는 형질 전환된 균주에서는 스쿠알렌 피크를 확인하였다(도 3b). 스쿠알렌 합성효소 유전자(trSQS 유전자), 데옥시자일룰로오스-5-포스페이트 합성효소 유전자(dxs 유전자) 디메틸알릴 디포스페이트 합성효소 유전자 (idi 유전자) 파네실 디포스페이트 합성 효소 (ispA 유전자)를 포함하는 제 1벡터와 sgRNA를 포함하지 않는 제 2벡터로 형질 전환 된 균주를 배양하여 배양 시작 후 12시간, 24시간, 36시간, 48시간, 60시간 동안 배양한 균주를 채취해 스쿠알렌을 생산량을 확인하였다. 그 결과, 도 4에서 보듯이, 각 시간별로 거의 일정한 스쿠알렌 생산량 을 보이는 것을 확인하였고, 4.5 mg/gDCW의 스쿠알렌을 생산하였다.
<실시예 5>
제1벡터 및 제2벡터에 의해 형질전환된 코리네박테리움 글루타미쿰 균주의 스쿠알렌 생산능
크리스퍼 유전자 조절 시스템 발현 벡터인 제2벡터(pCoryne-sgRNA -target-r)와 스쿠알렌을 생산균주의 제1벡터 (pBbEB1c-trSQS(CgCO) -dxs-idi-ispA)를 도입한 코리네박테리움 글루타미쿰 균주의 스쿠알렌 생산량을 확인하기 위해 다음과 같이 배양하였다. 배양조건은 30 ℃, 900 rpm의 바이오렉터를 이용한 배양으로 flower-48-micro well plate에 2% 글루코오스를 포함하는 CgXII 최소배지 1 mL 을 넣고 처음 배양시 OD 600을 1로 맞추어 접종해주었다. 배양 시 카나마이신 항생제 25 μg/mL와 클로람페니콜 항생제 7.5 μg/mL를 넣어주었으며, 유전자 발현을 위한 인듀서 1 mM IPTG, 100 nM aTc 를 첨가하였다. 배양 시작 후 48시간 동안 배양한 균주의 스쿠알렌 생산량을 측정하였다.
그 결과, trSQS 유전자, dxs 유전자, idi 유전자 ispA 유전자를 모두 갖는 제 1벡터와 sgRNA를 포함하지 않는 제 2벡터를 갖는 기존 스쿠알렌 생산 균주의 스쿠알렌 생산량 2.65 ± 0.034 mg/gDCW으로 측정되었다(도 5, control). 그에 비해 크리스퍼 유전자 저해 기술을 적용한 제2벡터로 형질전환된 균주에서는 스쿠알렌 생산량이 증가됨을 확인하였다(도 5). 즉, trSQS 유전자, dxs 유전자, idi 유전자 ispA 유전자를 모두 갖는 제 1벡터와 함께 크리스퍼 유전자 저해 기술을 적용한 균주의 스쿠알렌 생산량은 pCoryne-sgRNA-idsA-r1-dCas9 벡터를 포함하는 균주에서 3.47 ± 0.16 mg/gDCW 이고, pCoryne-sgRNAgdh-r1-dCas9 벡터를 포함하는 균주에서 3.60 ± 0.27 mg/gDCW 이며, pCoryne-sgRNAgapA-r1-dCas9 벡터를 포함하는 균주에서 4.28 ± 0.17 mg/gDCW로 측정되었다.
<실시예 6>
최적의 배지 조성
스쿠알렌을 생산하는 형질 전환된 코리네박테리움 글루타미쿰 균주에서 스쿠알렌 생산을 극대화시키기 위해 배지조건을 다양하게 적용하는 배지최적화 기법을 도입하였다. 균주의 배양은 바이오렉터를 이용하여 진행하였다. 배양조건은 30 ℃, 900 rpm이며 배지 조건은 flower-48-micro well plate에 2% 글루코오스를 포함하는 CgXII 최소배지를 기본으로 하고, 상기 배지의 성분 중 황산 암모늄을 0.01 g/L, 요소를 0.5 g/L로 질소 성분을 낮춘 배지(N) 1 mL, 2% 글루코오스를 포함하는 CgXII 최소배지에 15 g/L tryptone을 추가한 배지 1 mL 또는 2% 글루코오스를 포함하는 2YT (10 g/L yeast extract, 16 g/L tryptone, 5 g/L NaCl) 배지 1 mL 을 넣고 처음 배양 시 OD 600을 1로 맞추어 접종하였다. 배양시 카나마이신 항생제 25 μg/mL 와 클로람페니콜 항생제 7.5 μg/mL를 넣어주었으며, 유전자 발현을 위한 인듀서 1 mM IPTG, 100 nM aTc 를 첨가하였다.
배양 시작 후 48시간 동안 배양한 균주의 스쿠알렌 생산량을 측정하였다. CGXII 최소배지의 질소성분을 낮춘 배지 (CgXII N)를 적용한 경우 모든 균주에서 스쿠알렌 생산량이 증가했으며, 특히, 크리스퍼 유전자 저해 기술을 적용한 pCoryne-sgRNA-idsA-r1-dCas9 벡터를 포함하는 균주는 CgXII 최소배지에 tryptone을 추가한 배지와 2YT 배지에서 스쿠알렌 생산량이 향상되었다. [도 6]
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
한국미생물보존센터(국외) KCCM12189P 20171219
<110> Sungkyunkwan university <120> Transformed corynebacterium glutamicum having capability of producing squalene <130> PN1712-441 <160> 23 <170> KoPatentIn 3.0 <210> 1 <211> 1911 <212> DNA <213> Corynebacterium glutamicum <400> 1 atgggcattc tgaacagtat ttcaacacct gctgacttaa aggcccttaa tgatgaggat 60 ttggacgctc ttgccaaaga aatccgaact ttcctggtcg ataaagtcgc agcaactggt 120 ggccacttag gtccaaattt gggcgtagtg gaattaacca tcggtcttca tcgagttttc 180 gattcgcctc aagacccgat catctttgat acttctcacc agtcctatgt gcataagatc 240 ctgacgggtc gcgctaaaga ttttgattct ttgcgtcaaa aagatggcct ttctggttac 300 acctgccgtg ctgaaagtga gcacgattgg actgagtctt cgcatgcttc ggcggccttg 360 tcttatgcgg atggtttgtc taaagccaag cagttggatg gcgataccac gcatagtgtg 420 gttgctgtcg ttggtgatgg cgctctaact ggcggcatgt gttgggaagc actgaacaat 480 attgctgctg gtaaagaccg caaagttgtt gtcgtagtca atgacaatgg ccggagttat 540 tctccaacca ttggcggatt tgcggaaaac cttgcgggcc ttcgcatgca gcctttctat 600 gatcgcttca tggaaaaggg caagacgtcc ctgaaatcca tggggtgggt aggggagcgt 660 acttttgaag cgctccatgc atttaaagaa ggtgtgaaga gcaccgtcat tcccaccgaa 720 atgttccctg aactgggcat gaaatacgtg ggtccggttg atggacataa ccaaaaagct 780 gtcgacaatg cgctgaaata cgctcatgat tatgatggcc ccatcatcgt gcacatggtc 840 accgaaaagg gtcgtggtta cgcgcctgct gagcaggatt tggacgaatt gatgcactcc 900 acgggcgtca tcgatccgct cacaggagct cctaaatctg catcaaagcc cggttggacc 960 tctgtgttca gcgatgagct ggtcaagatt ggtgcgcaga atgaaaacgt tgttgccatc 1020 accgccgcga tggcaggtcc taccggtctg tccaagttcg aagccaattt ccccaaccga 1080 ttctttgatg tcggcattgc tgagcagcac gcggtaactt ctgccgcagg cctcgcattg 1140 ggtggaaaac accctgtggt ggctatttac tccacgttct tgaaccgcgc ttttgatcag 1200 ctgctcatgg atgtgggcat gctcaaccag cctgttactt tggtgcttga tcgctcaggt 1260 gtcacgggtt cggatggagc gagccacaat ggcgtctggg atatggcgct gacctcgatc 1320 gttccaggcg tgcaggtggc ggcaccacgt gatgaggatt ccttgcgtga gctgctcaat 1380 gaggctattt ccatcgatga tggccccaca gttgtgcgtt tccccaaggg cgacttgcca 1440 actccaattg ttgctatcga caccttggaa gacggcgtgg atgtcctcgc atatgaagac 1500 gccactgacg ttgaatcaac cgacgatgcg ccatcagttc tcatcattgc ggtaggcgag 1560 cgcgcaactg ttgcacttga cgttgcttcc aggattaaac agcacggcgt gaacgtcacg 1620 gttgttgacc cccgctggat tgtccccatc ccgcagtcct tggtcgcgct gtctgatgat 1680 catgacctcg tgatcaccat cgaagacggc gtcatccacg gcggcgtggg ctccttgctc 1740 tctgatgcgc ttaacgcctc tgaggtggat acccctcgcc gacaaatcgc cgtgccccag 1800 aagtacctgg atcacgcgtc ccgcaatgaa gtgctcgccg attatggcct cgacgccgac 1860 ggcattgaaa ccactgttgt tggatggctg gattccctgt tcggggaata a 1911 <210> 2 <211> 588 <212> DNA <213> Corynebacterium glutamicum <400> 2 gtgtctaagc ttaggggcat gactactgag gttgaactgg ttgttttagc tgattccgag 60 ggcaatccta ttggtactgc gccgaaagct acggtgcaca ctaaggacac gcctctgcat 120 ttcgcgtttt ccacctatat tttgaacccg cgtggggagc tgttggtgac gcgtcgtgca 180 ttgtcgaaga agacatggcc tggtgtgtgg acgaactcta tgtgtgggca ccctggtccg 240 gatgagacaa acgcggatgc gattcgtcgc aggggtgtcg atgagttggg gctggaggta 300 gattctttct tggatattca agagattctg cctgattacc agtaccgtgc tgtcgacgcg 360 tccggcattg tggagtggga gttgtgcccg gtccacctcg tgcgtttagc ggtgggggaa 420 tttgtggagc cactggatga tgaggtggag gagttcgagt gggcggaacc gcagaagctt 480 ttcgacgctg ttgatgccac accatttgtg ttttctccat ggctagtgga tcagcttagc 540 gcccctgagc tgcgccaagc catcctggaa gcgtttgacg cagagtaa 588 <210> 3 <211> 900 <212> DNA <213> Escherichia coli <400> 3 atggactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 60 tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 120 ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgttcggc 180 gttagcacaa acacgctgga cgcacccgct gccgccgttg agtgtatcca cgcttactca 240 ttaattcatg atgatttacc ggcaatggat gatgacgatc tgcgtcgcgg tttgccaacc 300 tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 360 gcgttctcga ttttaagcga tgccgatatg ccggaagtgt cggaccgcga cagaatttcg 420 atgatttctg aactggcgag cgccagtggt attgccggaa tgtgcggtgg tcaggcatta 480 gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 540 aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 600 ggacgtcgtg ctctgccggt actcgacaag tatgcagaga gcatcggcct tgccttccag 660 gttcaggatg acatcctgga tgtggtggga gatactgcaa cgttgggaaa acgccagggt 720 gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 780 aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 840 tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 900 900 <210> 4 <211> 1263 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgggcaagc tgctgcagct ggcactgcac ccagtggaaa tgaaggcagc actgaagctg 60 aagttctgcc gcaccccact gttctccatc tacgatcagt ccacctcccc atacctgctg 120 cactgcttcg aactgctgaa cctgacctcc cgctccttcg cagcagtgat ccgcgaactg 180 cacccagaac tgcgcaactg cgtgaccctg ttctacctga tcctgcgcgc actggatacc 240 atcgaagatg atatgtccat cgaacacgat ctgaagatcg atctgctgcg ccacttccac 300 gaaaagctgc tgctgaccaa gtggtccttc gatggcaacg caccagatgt gaaggatcgc 360 gcagtgctga ccgatttcga atccatcctg atcgagttcc acaagctgaa gccagaatac 420 caggaagtga tcaaggaaat caccgaaaag atgggcaacg gcatggcaga ttacatcctg 480 gatgaaaact acaacctgaa cggcctgcag accgtgcacg attacgatgt gtactgccac 540 tacgtggcag gcctggtggg cgatggcctg acccgcctga tcgtgatcgc aaagttcgca 600 aacgaatccc tgtactccaa cgaacagctg tacgaatcca tgggcctgtt cctgcagaag 660 accaacatca tccgcgatta caacgaagac ctggtggatg gccgctcctt ctggccaaag 720 gaaatctggt cccagtacgc accacagctg aaggatttca tgaagccaga aaacgaacag 780 ctgggcctgg attgcatcaa ccacctggtg ctgaacgcac tgtcccacgt gatcgatgtg 840 ctgacctacc tggcaggcat ccacgaacag tccaccttcc agttctgcgc aatcccacag 900 gtgatggcaa tcgcaaccct ggcactggtg ttcaacaacc gcgaagtgct gcacggcaac 960 gtgaagatcc gcaagggcac cacctgctac ctgatcctga agtcccgcac cctgcgcggc 1020 tgcgtggaaa tcttcgatta ctacctgcgc gatatcaagt ccaagctggc agtgcaggac 1080 ccaaacttcc tgaagctgaa catccagatt tccaagatcg aacagttcat ggaagaaatg 1140 taccaggata agctgccacc aaacgtgaag ccaaacgaaa ccccaatctt cctgaaggtg 1200 aaggaacgct cccgctacga tgatgaactg gtgccaaccc agcaggaaga agaatacaag 1260 taa 1263 <210> 5 <211> 10885 <212> DNA <213> Artificial Sequence <220> <223> pBbEB1c-trSQS(CgCO)-dxs-idi-ispA vector <400> 5 gacgtcgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa 60 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat 120 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg 180 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg 240 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc 300 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc 360 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac 420 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat 480 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac 540 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag 600 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc 660 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg 720 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg 780 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc 840 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga 900 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat 960 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg 1020 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc 1080 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 1140 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agcgcgaatt 1200 gatctggttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca 1260 gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct 1320 caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa 1380 atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg 1440 agcggataac aatttcagaa ttcaaaagat cttaatacgg acgaggttaa atagaaggag 1500 gtaatagatg ggcaagctgc tgcagctggc actgcaccca gtggaaatga aggcagcact 1560 gaagctgaag ttctgccgca ccccactgtt ctccatctac gatcagtcca cctccccata 1620 cctgctgcac tgcttcgaac tgctgaacct gacctcccgc tccttcgcag cagtgatccg 1680 cgaactgcac ccagaactgc gcaactgcgt gaccctgttc tacctgatcc tgcgcgcact 1740 ggataccatc gaagatgata tgtccatcga acacgatctg aagatcgatc tgctgcgcca 1800 cttccacgaa aagctgctgc tgaccaagtg gtccttcgat ggcaacgcac cagatgtgaa 1860 ggatcgcgca gtgctgaccg atttcgaatc catcctgatc gagttccaca agctgaagcc 1920 agaataccag gaagtgatca aggaaatcac cgaaaagatg ggcaacggca tggcagatta 1980 catcctggat gaaaactaca acctgaacgg cctgcagacc gtgcacgatt acgatgtgta 2040 ctgccactac gtggcaggcc tggtgggcga tggcctgacc cgcctgatcg tgatcgcaaa 2100 gttcgcaaac gaatccctgt actccaacga acagctgtac gaatccatgg gcctgttcct 2160 gcagaagacc aacatcatcc gcgattacaa cgaagacctg gtggatggcc gctccttctg 2220 gccaaaggaa atctggtccc agtacgcacc acagctgaag gatttcatga agccagaaaa 2280 cgaacagctg ggcctggatt gcatcaacca cctggtgctg aacgcactgt cccacgtgat 2340 cgatgtgctg acctacctgg caggcatcca cgaacagtcc accttccagt tctgcgcaat 2400 cccacaggtg atggcaatcg caaccctggc actggtgttc aacaaccgcg aagtgctgca 2460 cggcaacgtg aagatccgca agggcaccac ctgctacctg atcctgaagt cccgcaccct 2520 gcgcggctgc gtggaaatct tcgattacta cctgcgcgat atcaagtcca agctggcagt 2580 gcaggaccca aacttcctga agctgaacat ccagatttcc aagatcgaac agttcatgga 2640 agaaatgtac caggataagc tgccaccaaa cgtgaagcca aacgaaaccc caatcttcct 2700 gaaggtgaag gaacgctccc gctacgatga tgaactggtg ccaacccagc aggaagaaga 2760 atacaagtaa ggatctatta aagaggagaa tactagatgg gcattctgaa cagtatttca 2820 acacctgctg acttaaaggc ccttaatgat gaggatttgg acgctcttgc caaagaaatc 2880 cgaactttcc tggtcgataa agtcgcagca actggtggcc acttaggtcc aaatttgggc 2940 gtagtggaat taaccatcgg tcttcatcga gttttcgatt cgcctcaaga cccgatcatc 3000 tttgatactt ctcaccagtc ctatgtgcat aagatcctga cgggtcgcgc taaagatttt 3060 gattctttgc gtcaaaaaga tggcctttct ggttacacct gccgtgctga aagtgagcac 3120 gattggactg agtcttcgca tgcttcggcg gccttgtctt atgcggatgg tttgtctaaa 3180 gccaagcagt tggatggcga taccacgcat agtgtggttg ctgtcgttgg tgatggcgct 3240 ctaactggcg gcatgtgttg ggaagcactg aacaatattg ctgctggtaa agaccgcaaa 3300 gttgttgtcg tagtcaatga caatggccgg agttattctc caaccattgg cggatttgcg 3360 gaaaaccttg cgggccttcg catgcagcct ttctatgatc gcttcatgga aaagggcaag 3420 acgtccctga aatccatggg gtgggtaggg gagcgtactt ttgaagcgct ccatgcattt 3480 aaagaaggtg tgaagagcac cgtcattccc accgaaatgt tccctgaact gggcatgaaa 3540 tacgtgggtc cggttgatgg acataaccaa aaagctgtcg acaatgcgct gaaatacgct 3600 catgattatg atggccccat catcgtgcac atggtcaccg aaaagggtcg tggttacgcg 3660 cctgctgagc aggatttgga cgaattgatg cactccacgg gcgtcatcga tccgctcaca 3720 ggagctccta aatctgcatc aaagcccggt tggacctctg tgttcagcga tgagctggtc 3780 aagattggtg cgcagaatga aaacgttgtt gccatcaccg ccgcgatggc aggtcctacc 3840 ggtctgtcca agttcgaagc caatttcccc aaccgattct ttgatgtcgg cattgctgag 3900 cagcacgcgg taacttctgc cgcaggcctc gcattgggtg gaaaacaccc tgtggtggct 3960 atttactcca cgttcttgaa ccgcgctttt gatcagctgc tcatggatgt gggcatgctc 4020 aaccagcctg ttactttggt gcttgatcgc tcaggtgtca cgggttcgga tggagcgagc 4080 cacaatggcg tctgggatat ggcgctgacc tcgatcgttc caggcgtgca ggtggcggca 4140 ccacgtgatg aggattcctt gcgtgagctg ctcaatgagg ctatttccat cgatgatggc 4200 cccacagttg tgcgtttccc caagggcgac ttgccaactc caattgttgc tatcgacacc 4260 ttggaagacg gcgtggatgt cctcgcatat gaagacgcca ctgacgttga atcaaccgac 4320 gatgcgccat cagttctcat cattgcggta ggcgagcgcg caactgttgc acttgacgtt 4380 gcttccagga ttaaacagca cggcgtgaac gtcacggttg ttgacccccg ctggattgtc 4440 cccatcccgc agtccttggt cgcgctgtct gatgatcatg acctcgtgat caccatcgaa 4500 gacggcgtca tccacggcgg cgtgggctcc ttgctctctg atgcgcttaa cgcctctgag 4560 gtggataccc ctcgccgaca aatcgccgtg ccccagaagt acctggatca cgcgtcccgc 4620 aatgaagtgc tcgccgatta tggcctcgac gccgacggca ttgaaaccac tgttgttgga 4680 tggctggatt ccctgttcgg ggaataagga tctggatcta aagaggagaa atactaggtg 4740 tctaagctta ggggcatgac tactgaggtt gaactggttg ttttagctga ttccgagggc 4800 aatcctattg gtactgcgcc gaaagctacg gtgcacacta aggacacgcc tctgcatttc 4860 gcgttttcca cctatatttt gaacccgcgt ggggagctgt tggtgacgcg tcgtgcattg 4920 tcgaagaaga catggcctgg tgtgtggacg aactctatgt gtgggcaccc tggtccggat 4980 gagacaaacg cggatgcgat tcgtcgcagg ggtgtcgatg agttggggct ggaggtagat 5040 tctttcttgg atattcaaga gattctgcct gattaccagt accgtgctgt cgacgcgtcc 5100 ggcattgtgg agtgggagtt gtgcccggtc cacctcgtgc gtttagcggt gggggaattt 5160 gtggagccac tggatgatga ggtggaggag ttcgagtggg cggaaccgca gaagcttttc 5220 gacgctgttg atgccacacc atttgtgttt tctccatggc tagtggatca gcttagcgcc 5280 cctgagctgc gccaagccat cctggaagcg tttgacgcag agtaaggatc taagaaggag 5340 gtaaacaatg gactttccgc agcaactcga agcctgcgtt aagcaggcca accaggcgct 5400 gagccgtttt atcgccccac tgccctttca gaacactccc gtggtcgaaa ccatgcagta 5460 tggcgcatta ttaggtggta agcgcctgcg acctttcctg gtttatgcca ccggtcatat 5520 gttcggcgtt agcacaaaca cgctggacgc acccgctgcc gccgttgagt gtatccacgc 5580 ttactcatta attcatgatg atttaccggc aatggatgat gacgatctgc gtcgcggttt 5640 gccaacctgc catgtgaagt ttggcgaagc aaacgcgatt ctcgctggcg acgctttaca 5700 aacgctggcg ttctcgattt taagcgatgc cgatatgccg gaagtgtcgg accgcgacag 5760 aatttcgatg atttctgaac tggcgagcgc cagtggtatt gccggaatgt gcggtggtca 5820 ggcattagat ttagacgcgg aaggcaaaca cgtacctctg gacgcgcttg agcgtattca 5880 tcgtcataaa accggcgcat tgattcgcgc cgccgttcgc cttggtgcat taagcgccgg 5940 agataaagga cgtcgtgctc tgccggtact cgacaagtat gcagagagca tcggccttgc 6000 cttccaggtt caggatgaca tcctggatgt ggtgggagat actgcaacgt tgggaaaacg 6060 ccagggtgcc gaccagcaac ttggtaaaag tacctaccct gcacttctgg gtcttgagca 6120 agcccggaag aaagcccggg atctgatcga cgatgcccgt cagtcgctga aacaactggc 6180 tgaacagtca ctcgatacct cggcactgga agcgctagcg gactacatca tccagcgtaa 6240 taaataagga tccaaactcg agtaaggatc tccaggcatc aaataaaacg aaaggctcag 6300 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt 6360 cacactggct caccttcggg tgggcctttc tgcgtttata cctagggcgt tcggctgcgg 6420 cgagcggtat cagcagttat tggtgccctt cgaaatgacc gaccaagcga cgcccaacct 6480 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 6540 tttccgggac gccaacaaca agacccatca tagtttgccc ccgcgacatt gaccataaat 6600 tcatcgcaca aaatatcgaa cggggtttat gccgctttta gtgggtgcga agaatagtct 6660 gctcattacc cgcgaacacc gccgcattca gatcacgctt agtagcgtcc ccatgagtag 6720 gcagaaccgc gtccaagtcc acatcatcca taacgatcat gcacggggtg gaatccacac 6780 ccagacttgc cagcacctca ttagcgacac gttgcgcagc ggccacgtcc ttagccttat 6840 ccacgcaatc gagaacgtac tgcctaaccg cgaaatcaga ctgaatcagt ttccaatcat 6900 cgggcttcac caaagcaaca gcaacgcggg ttgattcgac ccgttccggt gcttccagac 6960 cggcgagctt gtacagttct tcttccattt cacgacgtac atcagcgtct atgtaatcaa 7020 tgcccaaagc acgcttagcc ccacgtgacc aggacgaacg caggttttta gaaccaacct 7080 catactcacg ccaccgagcc accaaaacag cgtccatatc ctcgccggcg tcgctttgat 7140 cggccaacat atccaacatc tgaaacggcg tgtacgaccc cttagacgcg gttttagtag 7200 cggagccagt cagttcctga gacatgccct tagcgaggta ggttgccatt ttcgcagcgt 7260 ctccacccca ggtagacacc tgatcaagtt tgaccccgtg ctcacgcagt ggcgcgtcca 7320 taccggcctt aaccacacca gcagaccagc gggaaaacat ggaatcctca aacgccttga 7380 gttcatcgtc agacagtgga cgatccaaga acaacagcat gttgcggtgc aagtgccaac 7440 cgttcgccca agagtctgtg acctcatagt cactataggt gtgctccacc ccgtaccgtg 7500 cacgttcttt cttccactga gatgttttca ccatcgaaga gtacgcagtc ttaatacccg 7560 cttcaacctg cgcaaatgac tgtgagcggt tgtgtcgaac agtgcccaca aacatcatga 7620 gcgcgccacc cgccgccaag tgattcttag tagcaatagc cagctcaatg cggcgttcgc 7680 ccatgacttc caattcagcc agaggtgacc cccagcgaga gtgagagttt tgcagaccct 7740 caaactgcga agcaccgtta gacgaccagg acaccgcaac agcttcgtcc ctgcgccacc 7800 tatggcaccc cgccagagcc ttactattgg tgatcttgta catgacgttt tgcctacgcc 7860 acgccctagc gcgagtgacc ttagaaccct cattgacctg cggttcctta gaggtgttca 7920 cttctatttc agtgttactc agtgttacct agacccgatg ttgtgcgggg ttgcgcagtg 7980 cgagtttgtg cgggtgttgt gcccgttgtc ttagctagtg ctatggttgt caattgaaac 8040 cccttcgggt tatgtggccc ccgtgcatat gagttggtag ctcgcacggg ggtttgtctt 8100 gtctagggac tattaatttt tagtggtgtt tggtggccgc ctagcttggc tatgcgtgcc 8160 agcttacccg tactcaatgt taaagatttg catcgacatg ggagggttac gtgtccgata 8220 cctagggggg gtatccgcga ctaggtgccc cggtgctcac tgtctgtacc ggcggggcaa 8280 gccccacacc ccgcatggac agggtggctc cgccccctgc acccccagca atctgcatgt 8340 acatgtttta cacattagca cgacatgact gcatgtgcat gcactgcatg cagactaggt 8400 aaatatgagt atgtacgact agtaacagga gcactgcaca taatgaatga gttgcaggac 8460 aatgtttgct acgcatgcgc atgacatatc gcaggaaagc tactagagtc ttaaagcatg 8520 gcaaccaagg cacagctaga acagcaacta caagaagctc aacaggcact acaggcgcag 8580 caagcgcagg cacaagccac catcgaagca ctagaagcgc aggcaaaggc taagcccgtc 8640 gtggtcaccg cacgcgttcc tttggcacta cgtgaggaca tgaagcgcgc aggcatgcag 8700 aacggtgaaa acctccaaga gttcatgatc gccgcgttta ccgagcggct agaaaagctc 8760 accaccaccg acaacgagga aaacaatgtc taacccacta gttctctttg cccaccgtga 8820 cccggtaaat gacgtgacgt tcgagtgcat tgagcacgcc acctacgaca cactttcaca 8880 cgctaaagac cagatcaccg cccaaatgca agccctagac gaagaagccg ccctactgcc 8940 ctaatgggtg tttcatgggt gtttccctag tgtttcatgg tgttttcacc taagctaggg 9000 aattgcgcga gaagtctcgc aaaaatcagc aacccccgga accacacagt tcacgggggt 9060 tcttctatgc cagaaatcag aaaggggaac cagtgaacga ccccgaatgg ctggatgatc 9120 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaaaaggat ctaggtgaag 9180 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 9240 tcagaccccg tatgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 9300 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 9360 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 9420 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 9480 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 9540 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 9600 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 9660 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 9720 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 9780 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 9840 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 9900 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 9960 attttggtca tgactagtgc ttggattctc accaataaaa aacgcccggc ggcaaccgag 10020 cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca acaggagtcc 10080 aagcgagctc gatatcaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt 10140 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca 10200 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga 10260 agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg 10320 ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt 10380 aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac 10440 tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac 10500 tatcccatat caccagctca ccgtctttca ttgccatacg aaattccgga tgagcattca 10560 tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg 10620 tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg 10680 actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc 10740 cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa 10800 atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat 10860 caacgtctca ttttcgccag atatc 10885 <210> 6 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-idsA-r1 <400> 6 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc tcatcacact 60 aagatacccg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 7 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-idsA-r3 <400> 7 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagta gacgtccttc 60 aatcgaatag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 8 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gapA-r1 <400> 8 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc aacctgtcct 60 caactttagg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 9 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gapA-r2 <400> 9 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtc gcaccaaacc 60 tactcacatg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 10 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gdh-r1 <400> 10 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtt aaatgctcac 60 gttcaattag ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 11 <211> 521 <212> RNA <213> Artificial Sequence <220> <223> sgRNA-gdh-r2 <400> 11 aattctaaag atctttgaca gctagctcag tcctaggtat aatactagtg agggcaccat 60 gtacatcctg ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact 120 tgaaaaagtg gcaccgagtc ggtgcttttt ttgaagcttg ggcccgaaca aaaactcatc 180 tcagaagagg atctgaatag cgccgtcgac catcatcatc atcatcattg agtttaaacg 240 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat 300 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc 360 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt 420 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa 480 gactgggcct ttcgttttat ctgttgtttg tcggtgaact g 521 <210> 12 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> iapA-r1 promotor <400> 12 ctcatcacac taagataccc 20 <210> 13 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> iapA-r3 promotor <400> 13 agacgtcctt caatcgaata 20 <210> 14 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gapA-r1 promotor <400> 14 caacctgtcc tcaactttag 20 <210> 15 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gapA-r2 promotor <400> 15 cgcaccaaac ctactcacat 20 <210> 16 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gdh-r1 promotor <400> 16 taaatgctca cgttcaatta 20 <210> 17 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> gdh-r2 promotor <400> 17 gagggcacca tgtacatcct 20 <210> 18 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-idsA-r1-dCas9 vector <400> 18 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtctca tcacactaag atacccgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060 <210> 19 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-idsA-r3-dCas9 vector <400> 19 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtagac gtccttcaat cgaatagttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060 <210> 20 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gapA-r1-dCas9 vector <400> 20 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtcaac ctgtcctcaa ctttaggttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060 <210> 21 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gapA-r2-dCas9 vector <400> 21 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtcgca ccaaacctac tcacatgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060 <210> 22 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gdh-r1-dCas9 vector <400> 22 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagttaaa tgctcacgtt caattagttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060 <210> 23 <211> 12060 <212> DNA <213> Artificial Sequence <220> <223> pCoryne-sgRNA-gdh-r2-dCas9 vector <400> 23 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 60 tatttcgttc atccatagtt gcctgagaat tctaaagatc tttgacagct agctcagtcc 120 taggtataat actagtgagg gcaccatgta catcctgttt tagagctaga aatagcaagt 180 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg 240 aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat 300 catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc ggatgagaga 360 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 420 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 480 gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 540 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 600 gtgaactgga tccttaagac ccactttcac atttaagttg tttttctaat ccgcatatga 660 tcaattcaag gccgaataag aaggctggct ctgcaccttg gtgatcaaat aattcgatag 720 cttgtcgtaa taatggcggc atactatcag tagtaggtgt ttccctttct tctttagcga 780 cttgatgctc ttgatcttcc aatacgcaac ctaaagtaaa atgccccaca gcgctgagtg 840 catataatgc attctctagt gaaaaacctt gttggcataa aaaggctaat tgattttcga 900 gagtttcata ctgtttttct gtaggccgtg tacctaaatg tacttttgct ccatcgcgat 960 gacttagtaa agcacatcta aaacttttag cgttattacg taaaaaatct tgccagcttt 1020 ccccttctaa agggcaaaag tgagtatggt gcctatctaa catctcaatg gctaaggcgt 1080 cgagcaaagc ccgcttattt tttacatgcc aatacaatgt aggctgctct acacctagct 1140 tctgggcgag tttacgggtt gttaaacctt cgattccgac ctcattaagc agctctaatg 1200 cgctgttaat cactttactt ttatctaatc tagacatcat taattcctaa tttttgttga 1260 cactctatcg ttgatagagt tattttacca ctccctatca gtgatagaga ttgacatccc 1320 tatcagtgat agagatactg agcacaacat ctgatctaaa gaggagaaag gatctatgga 1380 taagaagtac tccatcggcc tggcaatcgg caccaactcc gtgggctggg cagtgatcac 1440 cgatgaatac aaggtgccat ccaagaagtt caaggtgctg ggcaacaccg atcgccactc 1500 catcaagaag aacctgatcg gcgcactgct gttcgattcc ggcgaaaccg cagaagcaac 1560 ccgcctgaag cgcaccgcac gccgccgcta cacccgccgc aagaaccgca tctgctacct 1620 ccaggaaatc ttctccaacg aaatggcaaa ggtggatgat tccttcttcc accgcctgga 1680 agaatccttc ctggtggaag aagataagaa gcacgaacgc cacccaatct tcggcaacat 1740 cgtggatgaa gtggcatacc acgaaaagta cccaaccatc taccacctgc gcaagaagct 1800 ggtggattcc accgataagg cagacctgcg cctgatctac ctggcactgg cacacatgat 1860 caagttccgc ggccacttcc tgatcgaagg cgatctgaac ccagataact ccgatgtgga 1920 taagctgttc atccagctgg tgcagaccta caaccagctg ttcgaagaaa acccaatcaa 1980 cgcatccggc gtggatgcaa aggcaatcct gtccgcacgc ctgtccaagt cccgccgcct 2040 ggaaaacctg atcgcacagc tgccaggcga aaagaagaac ggcctgttcg gcaacctgat 2100 cgcactgtcc ctgggcctga ccccaaactt caagtccaac ttcgatctgg cagaagatgc 2160 aaagctccag ctgtccaagg atacctacga tgatgatctg gataacctgc tggcacagat 2220 cggcgatcag tacgcagacc tgttcctggc agcaaagaac ctgtccgatg caatcctgct 2280 gtccgatatc ctgcgcgtga acaccgaaat caccaaggca ccactgtccg catccatgat 2340 caagcgctac gatgaacacc accaggatct gaccctgctg aaggcactgg tgcgccagca 2400 gctgccagaa aagtacaagg aaatcttctt cgatcagtcc aagaacggct acgcaggcta 2460 catcgatggc ggcgcatccc aggaagagtt ctacaagttc atcaagccaa tcctggaaaa 2520 gatggatggc accgaagaac tgctggtgaa gctgaaccgc gaagacctgc tgcgcaagca 2580 gcgcaccttc gataacggct ccatcccaca ccagatccac ctgggcgaac tgcacgcaat 2640 cctgcgccgc caggaagatt tctacccatt cctgaaggat aaccgcgaaa agatcgaaaa 2700 gatcctgacc ttccgcatcc catactacgt gggcccactg gcacgcggca actcccgctt 2760 cgcatggatg acccgcaagt ccgaagaaac catcacccca tggaacttcg aagaagtggt 2820 ggataagggc gcatccgcac agtccttcat cgaacgcatg accaacttcg ataagaacct 2880 gccaaacgaa aaggtgctgc caaagcactc cctgctgtac gaatacttca ccgtgtacaa 2940 cgaactgacc aaggtgaagt acgtgaccga aggcatgcgc aagccagcat tcctgtccgg 3000 cgaacagaag aaggcaatcg tggatctgct gttcaagacc aaccgcaagg tgaccgtgaa 3060 gcagctgaag gaagattact tcaagaagat cgaatgcttc gattccgtgg aaatctccgg 3120 cgtggaagat cgcttcaacg catccctggg cacctaccac gatctgctga agatcatcaa 3180 ggataaggat ttcctggata acgaagaaaa cgaagatatc ctggaagata tcgtgctgac 3240 cctgaccctg ttcgaagatc gcgaaatgat cgaagaacgc ctgaagacct acgcacacct 3300 gttcgatgat aaggtgatga agcagctgaa gcgccgccgc tacaccggct ggggccgcct 3360 gtcccgcaag ctgatcaacg gcatccgcga taagcagtcc ggcaagacca tcctggattt 3420 cctgaagtcc gatggcttcg caaaccgcaa cttcatgcag ctgatccacg atgattccct 3480 gaccttcaag gaagatatcc agaaggcaca ggtgtccggc cagggcgatt ccctgcacga 3540 acacatcgca aacctggcag gctccccagc aatcaagaag ggcatcctcc agaccgtgaa 3600 ggtggtggat gaactggtga aggtgatggg ccgccacaag ccagaaaaca tcgtgatcga 3660 aatggcacgc gaaaaccaga ccacccagaa gggccagaag aactcccgcg aacgcatgaa 3720 gcgcatcgaa gaaggcatca aggaactggg ctcccagatc ctgaaggaac acccagtgga 3780 aaacacccag ctccagaacg aaaagctgta cctgtactac ctccagaacg gccgcgatat 3840 gtacgtggat caggaactgg atatcaaccg cctgtccgat tacgatgtgg atgcaatcgt 3900 gccacagtcc ttcctgaagg atgattccat cgataacaag gtgctgaccc gctccgataa 3960 gaaccgcggc aagtccgata acgtgccatc cgaagaagtg gtgaagaaga tgaagaacta 4020 ctggcgccag ctgctgaacg caaagctgat cacccagcgc aagttcgata acctgaccaa 4080 ggcagaacgc ggcggcctgt ccgaactgga taaggcaggc ttcatcaagc gccagctggt 4140 ggaaacccgc cagatcacca agcacgtggc acagatcctg gattcccgca tgaacaccaa 4200 gtacgatgaa aacgataagc tgatccgcga agtgaaggtg atcaccctga agtccaagct 4260 ggtgtccgat ttccgcaagg atttccagtt ctacaaggtg cgcgaaatca acaactacca 4320 ccacgcacac gatgcatacc tgaacgcagt ggtgggcacc gcactgatca agaagtaccc 4380 aaagctggaa tccgagttcg tgtacggcga ttacaaggtg tacgatgtgc gcaagatgat 4440 cgcaaagtcc gaacaggaaa tcggcaaggc aaccgcaaag tacttcttct actccaacat 4500 catgaacttc ttcaagaccg aaatcaccct ggcaaacggc gaaatccgca agcgcccact 4560 gatcgaaacc aacggcgaaa ccggcgaaat cgtgtgggat aagggccgcg atttcgcaac 4620 cgtgcgcaag gtgctgtcca tgccacaggt gaacatcgtg aagaagaccg aagtgcagac 4680 cggcggcttc tccaaggaat ccatcctgcc aaagcgcaac tccgataagc tgatcgcacg 4740 caagaaggat tgggacccaa agaagtacgg cggcttcgat tccccaaccg tggcatactc 4800 cgtgctggtg gtggcaaagg tggaaaaggg caagtccaag aagctgaagt ccgtgaagga 4860 actgctgggc atcaccatca tggaacgctc ctccttcgaa aagaacccaa tcgatttcct 4920 ggaagcaaag ggctacaagg aagtgaagaa ggatctgatc atcaagctgc caaagtactc 4980 cctgttcgaa ctggaaaacg gccgcaagcg catgctggca tccgcaggcg aactccagaa 5040 gggcaacgaa ctggcactgc catccaagta cgtgaacttc ctgtacctgg catcccacta 5100 cgaaaagctg aagggctccc cagaagataa cgaacagaag cagctgttcg tggaacagca 5160 caagcactac ctggatgaaa tcatcgaaca gatttccgag ttctccaagc gcgtgatcct 5220 ggcagatgca aacctggata aggtgctgtc cgcatacaac aagcaccgcg ataagccaat 5280 ccgcgaacag gcagaaaaca tcatccacct gttcaccctg accaacctgg gcgcaccagc 5340 agcattcaag tacttcgata ccaccatcga tcgcaagcgc tacacctcca ccaaggaagt 5400 gctggatgca accctgatcc accagtccat caccggcctg tacgaaaccc gcatcgatct 5460 gtcccagctg ggcggcgatt aactgcagcc aagcttctgt tttggcggat gagagaagat 5520 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 5580 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 5640 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa 5700 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 5760 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 5820 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 5880 ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt tttctaaata 5940 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 6000 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 6060 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 6120 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 6180 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 6240 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct 6300 cagaatgact tggttgagta ctgcggcgtc gctgatcgcc ctggcgacgt tgtgcgggtg 6360 gcttgtccct gagggcgctg cgacagatag ctaaaaatct gggtcaggat cgccgtagag 6420 cgcgcgtcgt cgattggagg cttccccttt ggttgacggt cttcaatcgc tctacggcga 6480 tcctgacgct tttttgttgc gtaccgtcga tcgttttatt tctgtcgatc ccgaaaaagt 6540 ttttgccttt tgtaaaaaac ttctcggtcg ccccgcaaat tttcgattcc agatttttta 6600 aaaaccaagc cagaaatacg acacaccgtt tgcagataat ctgtctttcg gaaaaatcaa 6660 gtgcgataca aaatttttag cacccctgag ctgcgcaaag tcccgcttcg tgaaaatttt 6720 cgtgccgcgt gattttccgc caaaaacttt aacgaacgtt cgttataatg gtgtcatgac 6780 cttcacgacg aagtactaaa attggcccga atcatcagct atggatctct ctgatgtcgc 6840 gctggagtcc gacgcgctcg atgctgccgt cgatttaaaa acggtgatcg gatttttccg 6900 agctctcgat acgacggacg cgccagcatc acgagactgg gccagtgccg cgagcgacct 6960 agaaactctc gtggcggatc ttgaggagct ggctgacgag ctgcgtgctc ggccagcgcc 7020 aggaggacgc acagtagtgg aggatgcaat cagttgcgcc tactgcggtg gcctgattcc 7080 tccccggcct gacccgcgag gacggcgcgc aaaatattgc tcagatgcgt gtcgtgccgc 7140 agccagccgc gagcgcgcca acaaacgcca cgccgaggag ctggaggcgg ctaggtcgca 7200 aatggcgctg gaagtgcgtc ccccgagcga aattttggcc atggtcgtca cagagctgga 7260 agcggcagcg agaattatcc gcgatcgtgg cgcggtgccc gcaggcatga caaacatcgt 7320 aaatgccgcg tttcgtgtgg ccgtggccgc ccaggacgtg tcagcgccgc caccacctgc 7380 accgaatcgg cagcagcgtc gcgcgtcgaa aaagcgcaca ggcggcaaga agcgataagc 7440 tgcacgaata cctgaaaaat gttgaacgcc ccgtgagcgg taactcacag ggcgtcggct 7500 aacccccagt ccaaacctgg gagaaagcgc tcaaaaatga ctctagcgga ttcacgagac 7560 attgacacac cggcctggaa attttccgct gatctgttcg acacccatcc cgagctcgcg 7620 ctgcgatcac gtggctggac gagcgaagac cgccgcaaat tcctcgctca cctgggcaga 7680 gaaaatttcc agggcagcaa gacccgcgac ttcgccagcg cttggatcaa agacccggac 7740 acgggagaaa cacagccgaa gttataccga gttggttcaa aatcgcttgc ccggtgccag 7800 tatgttgctc tgacgcacgc gcagcacgca gccgtgcttg tcctggacat tgatgtgccg 7860 agccaccagg ccggcgggaa aatcgagcac gtaaaccccg aggtctacgc gattttggag 7920 cgctgggcac gcctggaaaa agcgccagct tggatcggcg tgaatccact gagcgggaaa 7980 tgccagctca tctggctcat tgatccggtg tatgccgcag caggcatgag cagcccgaat 8040 atgcgcctgc tggctgcaac gaccgaggaa atgacccgcg ttttcggcgc tgaccaggct 8100 ttttcacata ggctgagccg tggccactgc actctccgac gatcccagcc gtaccgctgg 8160 catgcccagc acaatcgcgt ggatcgccta gctgatctta tggaggttgc tcgcatgatc 8220 tcaggcacag aaaaacctaa aaaacgctat gagcaggagt tttctagcgg acgggcacgt 8280 atcgaagcgg caagaaaagc cactgcggaa gcaaaagcac ttgccacgct tgaagcaagc 8340 ctgccgagcg ccgctgaagc gtctggagag ctgatcgacg gcgtccgtgt cctctggact 8400 gctccagggc gtgccgcccg tgatgagacg gcttttcgcc acgctttgac tgtgggatac 8460 cagttaaaag cggctggtga gcgcctaaaa gacaccaagg gtcatcgagc ctacgagcgt 8520 gcctacaccg tcgctcaggc ggtcggagga ggccgtgagc ctgatctgcc gccggactgt 8580 gaccgccaga cggattggcc gcgacgtgtg cgcggctacg tcgctaaagg ccagccagtc 8640 gtccctgctc gtcagacaga gacgcagagc cagccgaggc gaaaagctct ggccactatg 8700 ggaagacgtg gcggtaaaaa ggccgcagaa cgctggaaag acccaaacag tgagtacgcc 8760 cgagcacagc gagaaaaact agctaagtcc agtcaacgac aagctaggaa agctaaagga 8820 aatcgcttga ccattgcagg ttggtttatg actgttgagg gagagactgg ctcgtggccg 8880 acaatcaatg aagctatgtc tgaatttagc gtgtcacgtc agaccgtgaa tagagcactt 8940 aaggtctgcg ggcattgaac ttccacgagg acgccgaaag cttcccagta aatgtgccat 9000 ctcgtaggca gaaaacggtt cccccgtagg gtctctctct tggcctcctt tctaggtcgg 9060 gctgattgct cttgaagctc tctagggggg ctcacaccat aggcagataa cgttccccac 9120 cggctcgcct cgtaagcgca caaggactgc tcccaaagat cttcaaagcc actgccgcga 9180 ctgccttcgc gaagccttgc cccgcggaaa tttcctccac cgagttcgtg cacaccccta 9240 tgccaagctt ctttcaccct aaattcgaga gattggattc ttaccgtgga aattcttcgc 9300 aaaaatcgtc ccctgatcgc ccttgcgacg ttggcgtcgg tgccgctggt tgcgcttggc 9360 ttgaccgact tgatcctccg gcgttcagcc tgtgccacag ccgacaggat ggtgaccacc 9420 atttgcccca tatcaccgtc ggtactgatc ccgtcgtcaa taaaccgaac cgctacaccc 9480 tgagcatcaa actcttttat cagttggatc atgtcggcgt gtcgcggcca agacggtcga 9540 gcttcttcac cagaatgaca tcaccttcct ccaccttcat cctcagcaaa tccagccctt 9600 cccgatctgt tgaactgccg gatgccttgt cggtaaagat gcggttagct tttacccctg 9660 catctttgag cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 9720 aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 9780 ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 9840 aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 9900 tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 9960 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 10020 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 10080 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 10140 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 10200 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 10260 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 10320 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 10380 aaccggcgca ggaacactgc cagcgcatca gcaatatttt cacctgaatc aggatattct 10440 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 10500 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 10560 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 10620 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 10680 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 10740 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 10800 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 10860 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag 10920 atcacgcatc ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc 10980 aactggtcca cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat 11040 gatggggcga ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag 11100 cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc 11160 ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat 11220 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga 11280 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga 11340 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac 11400 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg 11460 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc 11520 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc 11580 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt 11640 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc 11700 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa 11760 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt 11820 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc 11880 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg 11940 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 12000 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 12060 12060

Claims (22)

  1. 피루베이트(pyruvate) 및 D-글리세르알데하이드 3-포스페이트(D-glyceraldehyde 3-phosphate, G3P)로부터 1-데옥시-D-크실룰로오스 5-포스페이트(1-deoxy-D-xylulose 5-phosphate, DXP)를 생산하는 효소인 데옥시크실룰로오스-5-포스페이트 합성효소 (deoxyxylulose-5-phosphate synthase, dxs)를 코딩하는유전자;
    아이소펜테닐 디포스페이트(isophentenyl diphosphate, IPP)로부터 디메틸알릴 디포스페이트(dimethylallyl diphosphate, DMAPP)를 생산하는 효소인 아이소펜테닐-디포스페이트 델타 아이소머라아제(isopentenyl diphosphate delta isomerase, idi)를 코딩하는 유전자;
    디메틸알릴 디포스페이트(DMAPP)로부터 파네실 디포스페이트(farnesyl diphosphate, FPP)를 생산하는 효소인 제라닐트랜스퍼라아제(geranyl transferase, ispA)를 코딩하는 유전자; 및
    파네실 디포스페이트(FPP)로부터 스쿠알렌(squalene)을 생산하는 효소인 스쿠알렌 합성 효소를 코딩하는 유전자를 포함하는 코리네박테리움 글루타미쿰 균주.
  2. 제1항에 있어서, 상기 데옥시크실룰로오스-5-포스페이트 합성효소를 코딩하는 유전자는, 서열번호 1의 염기서열을 포함하고,
    아이소펜테닐-디포스페이트 델타 아이소머라아제를 코딩하는 유전자는 서열번호 2의 염기서열을 포함하고,
    제라닐 트랜스퍼라아제를 코딩하는 유전자는 서열번호 3의 염기서열을 포함하며,
    스쿠알렌 합성 효소를 코딩하는 유전자는 서열번호 4의 염기 서열을 포함하는, 코리네박테리움 글루타미쿰 균주.
  3. 삭제
  4. 제1항에 있어서, 상기 데옥시크실룰로오스-5-포스페이트 합성효소 (deoxyxylulose-5-phosphate synthase, dxs) 유전자 및 아이소펜테닐-디포스페이트 델타 아이소머라아제(isopentenyl diphosphate delta isomerase, idi) 유전자는 코리네박테리움 글루타미쿰으로부터 유래한 것인, 코리네박테리움 글루타미쿰 균주.
  5. 제1항에 있어서, 제라닐트랜스퍼라아제(geranyl transferase, ispA) 유전자는 대장균(E.coli)로부터 유래된 것인, 코리네박테리움 글루타미쿰 균주.
  6. 제1항에 있어서, 스쿠알렌 합성효소 유전자는 사카로마이세스 세레비지에(Saccharomyces cerevisiae)로부터 유래된 것인, 코리네박테리움 글루타미쿰 균주.
  7. 제1항에 있어서, 상기 균주는 dxs 유전자, idi 유전자, ispA 유전자 및 스쿠알렌 합성효소 유전자로부터 유래된 염기서열을 포함하는 벡터로 형질 전환된 것을 특징으로 하는 코리네박테리움 글루타미쿰 균주.
  8. 제1항에 있어서,
    상기 균주는 서열번호 6 내지 서열번호 11의 염기서열 중에서 선택되는 유전자의 발현을 저해하는 sgRNA를 포함하는 각각의 벡터 중 1 이상 선택된 벡터를 추가적으로 포함하여 형질 전환된 것을 특징으로 하는 코리네박테리움 글루타미쿰 균주.
  9. 제8항에 있어서, 상기 sgRNA는 서열번호 12 내지 서열번호 17의 프라이머로부터 선택되는 1 이상의 프라이머로부터 제작되는 것을 특징으로 하는 코리네박테리움 글루타미쿰 균주.
  10. 삭제
  11. 제8항에 있어서, 상기 벡터는 dCas9 단백질을 코딩하는 유전자를 포함하는 것을 특징으로 하는, 코리네박테리움 글루타미쿰 균주.
  12. 제8항에 있어서, 상기 유전자의 발현 저해는 크리스퍼 유전자 조절 시스템에 의한 것임을 특징으로 하는, 코리네박테리움 글루타미쿰 균주.
  13. 제1항에 있어서,
    상기 균주는 제1벡터 및 제2벡터로 형질전환된 것으로서,
    상기 제1벡터는, dxs 유전자, idi 유전자, ispA 유전자 및 스쿠알렌 합성효소 유전자를 포함하고,
    상기 제2벡터는 서열번호 6 내지 서열번호 11의 염기서열 중에서 선택된 유전자의 발현을 저해하는 sgRNA 및 dCas9 유전자를 포함하는 각각의 벡터로부터 1이상 선택되는 것을 특징으로 하는 코리네박테리움 글루타미쿰 균주.
  14. 제13항에 있어서,
    상기 제1벡터는 서열번호 5의 서열인 것을 특징으로 하는, 코리네박테리움 글루타미쿰 균주.
  15. 삭제
  16. 제13항에 있어서,
    상기 제2벡터는 서열번호 18 내지 서열번호 23의 서열 중 하나인 것을 특징으로 하는, 코리네박테리움 글루타미쿰 균주.
  17. 제1항에 있어서, 상기 균주는, 기탁번호 KCCM12189P 인 코리네박테리움 글루타미쿰 균주.
  18. 제1항 내지 제2항, 제4항 내지 제9항, 제11항 내지 제14항 및 제16항 내지 제17항 중 어느 한 항의 균주를 배양하는 단계를 포함하는, 스쿠알렌 생산 방법.
  19. 제18항에 있어서,
    상기 균주는 황산 암모늄 0.001 내지 0.01 g/L 및 요소 0.01 내지 0.5 g/L를 포함하는 CgXII 최소 배지를 포함하는 배양액을 이용하여 배양하는 것임을 특징으로 하는, 스쿠알렌 생산 방법.
  20. 제19항에 있어서,
    상기 배양액에 트립톤을 추가로 포함하는 것을 특징으로 하는, 스쿠알렌 생산 방법.
  21. 제20항에 있어서,
    상기 트립톤은 10 내지 20g/L로 포함하는 것을 특징으로 하는 스쿠알렌 생산 방법.
  22. 제19항에 있어서,
    상기 배양액에 이스트 추출물 1g/L 내지 20g/L, 염화나트륨 1g/L 내지 10g/L을 추가로 포함하는 것을 특징으로 하는 스쿠알렌 생산 방법.
KR1020180005193A 2018-01-15 2018-01-15 스쿠알렌 생성능을 가지는 형질전환된 코리네박테리움 글루타미쿰 KR102081699B1 (ko)

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