LOCUS Exported 6172 bp ds-DNA circular SYN 26-JUL-2022 DEFINITION CTLA4 gene knockin with mutation. ACCESSION . VERSION . KEYWORDS pIG-719 CTLA4-GFP R70W SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6172) AUTHORS Shy BR, Vykunta V, Ha A, Roth TL, Talbot A, Nguyen DN, Chen YY, Blaeschke F, Vedova S, Mamedov MR, Chung J, Li H, Wolf J, Martin TG, Ye L, Eyquem J, Esensten JH, Alexander M TITLE Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing JOURNAL bioRxiv 2021.09.02.458799 REFERENCE 2 (bases 1 to 6172) AUTHORS . TITLE Direct Submission JOURNAL Exported Jul 26, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6172 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(47..69) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 169..188 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 382..404 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 396..413 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 397..413 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" CDS 2061..2519 /codon_start=1 /product="improved N-terminal fragment of mVenus for use in bimolecular fluorescence complementation (BiFC) (Kodama and Hu, 2010)" /label=VN155(I152L) /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA" primer_bind complement(2100..2121) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 2708..2729 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2763..2884 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2800..2819) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2854..2873 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(3969..3985) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3969..3985) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(3982..4004) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 3993..4009 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(4017..4047) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4062..4083 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4200..4217) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4371..4959) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4451..4470) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5130..5990) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 5753..5772 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5991..6095) /gene="bla" /label=AmpR promoter primer_bind 6163..9 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" ORIGIN 1 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 61 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 121 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 181 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa 241 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg 301 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa 361 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcc 421 acagtgctaa ggatgctcag agggcatctc tgataggcag aggtgagggt tagggaagga 481 agctgtagtc tagctagcta gagctgctgg aatagacatg acaatggctg ctgccaaact 541 gttttctctt ctgaggacag atgtcccgtg caagtggctt ggtggaaggg actagtgtct 601 ctaatatagg gtgatttata agcaggaaag tgtgtcctag aaattcagac cagagtgata 661 gattggaatt ggatcatggg ggactcattg aatgttattt attgtatttg tttttgcgat 721 cagtgttagt aaagtgtcaa agggattgag cagatgagtg acatcatgca acacaagttt 781 tgagtttcac ttgtcagact gactggagag gggcctggtt agttacagga aggtaatttg 841 gcatgcagcc actatttttg agttgatgca agcctctctg tatggagagc tggtctcctt 901 tatcctgtgg gaaaagagaa caaaggagca tgggagtgtt caagggaagg agaaataaag 961 ggcagagagg cagcggtggt gtcaggggaa gcccacagga gttaacagca gggttgcctc 1021 aacctagaga ggaagcgacc tggtgccctc ggctctgtgg cttccttcat ctaacaacat 1081 cttccactct acaacaatgc cagggaaggc ggaggctggt acagtgcatc aagacacagc 1141 tactcctggg tgacagaggt tcagggccag ctcactaagt aggcagaagt ttttgacata 1201 tactttgaga gataaagcaa gattctgtac ctcaaccttc agaatttccc ctaccactca 1261 ttatagttcc ggagctatat agctcctatc attctatcat aaccttagaa taccagagaa 1321 catatcatct catctaatta tctcttacta tatgtgaaaa aaatgaagga catgggggaa 1381 gtgtgacttg ccccaaatca catatttcat ggtagagggc tgggcttggc catgaaggag 1441 catgagttca ctgagttccc tttggctttt ccatgctagc aatgcacgtg gcccagcctg 1501 ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat gcatctccag 1561 gcaaagccac tgaggtctgg gtgacagtgc ttcggcaggc tgacagccag gtgactgaag 1621 tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat tccatctgca 1681 cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg gccatggaca 1741 cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac ctgggcatag 1801 gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct gacttcctcc 1861 tctggatcct tgcagcagtt agttcggggt tgttttttta tagctttctc ctcacagctg 1921 tttctttgag caaaatgcta aagaaaagaa gccctcttac aacaggggtc tatgtgaaaa 1981 tgcccccaac agagccagaa tgtgaaaagc aatttcagcc ttattttatt cccatcaatg 2041 gatctggagg aactagcggc agcaagggcg aggagctgtt caccggggtg gtgcccatcc 2101 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg 2161 gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 2221 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 2281 ccgaccacat gaagcgccac gacttcttca agtccgccat gcccgaaggc tacgtccagg 2341 agcgcaccat cagcttcaag gacgacggca cctacaagac ccgcgccgag gtgaagttcg 2401 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 2461 acatcctggg gcacaagctg gagtacaact tcaacagcca caacgtctat atcaccgccg 2521 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacgtg gaggacggca 2581 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 2641 tgcccgacaa ccactacctg agcacccagt ccgtgctgag caaagacccc aacgagaagc 2701 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcacttga caccgggtct 2761 tcaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 2821 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 2881 cttatcatgt ctggaagacc tgtttacctc ttctgtttgt catatcagtg ttcttcctgc 2941 cacaaccatc ttgaagaatc tatttctcag taagaaaata tctttatgga gagtagctgg 3001 aaaacagttg agagatggag gggaggctgg gggtgtggag aggggaaggg gtaagtgata 3061 gattcgttga aggggggaga aaaggccgtg gggatgaagc tagaaggcag aagggcttgc 3121 ctgggcttgg ccatgaagga gcatgagttc actgagttcc ctttggcttt tccatgctac 3181 caatgcacgt ggcccagcct gctgtggtac tggccagcag ccgaggcatc gccagctttg 3241 tgtgtgagta tgcatctcca ggcaaagcca ctgaggtccg ggtgacagtg cttcggcagg 3301 ctgacagcca ggtgactgaa gtctgtgcgg caacctacat gatggggaat gagttgacct 3361 tcctagatga ttccatctgc acgggcacct ccagtggaaa tcaagtgaac ctcactatcc 3421 aaggactgag ggccatggac acgggactct acatctgcaa ggtggagctc atgtacccac 3481 cgccatacta cctgggcata ggcaacggaa cccagattta tgtaattggt gagcaaagcc 3541 atttcactga gttgacacct gttgcattgc agtcatctat gcacaaaaac agttttgttc 3601 cttaatttca ggaggtttac ttttaggact gtggacattc tctttaagag ttctgtacca 3661 catggtagcc ttgcttattg tgggtggcaa ccttaatagc attctgactg taaaataaaa 3721 tgatttgggg aagttggggc tctcgctctg gagtgctaac catcatgacg tttgatctgt 3781 acttttgata tgatatgatg ctcctgggga agtagtccca aatagccaaa cctattggtg 3841 ggctacccat gcaatttagg ggtggacctc aaggcctgga agctctaatg tccttttttc 3901 accaatgttg gggagtagag ccctagtcta gagtcgacct gcaggcatgc aagcttggcg 3961 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 4021 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 4081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 4141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 4201 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 4261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 4321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 4381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 4441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 4501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 4561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 4621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 4681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 4741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 4801 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 4861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 4921 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 4981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 5041 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 5101 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 5161 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 5221 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 5281 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 5341 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 5401 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 5461 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 5521 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 5581 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 5641 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 5701 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 5761 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 5821 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 5881 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 5941 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 6001 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 6061 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 6121 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg ta //