LOCUS       sequence-367836-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CTLA4 gene knockin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  AUTHORS   Shy BR, Vykunta VS, Ha A, Talbot A, Roth TL, Nguyen DN, Pfeifer WG, 
            Chen YY, Blaeschke F, Shifrut E, Vedova S, Mamedov MR, Chung JJ, Li 
            H, Yu R, Wu D, Wolf J, Martin TG, Castro CE, Ye L, Esensten JH, 
            Eyquem J, Marson A
  TITLE     High-yield genome engineering in primary cells using a hybrid ssDNA 
            repair template and small-molecule cocktails.
  JOURNAL   Nat Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8.
  PUBMED    36008610
REFERENCE   2  (bases 1 to 6172)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2022 Aug 25:10.1038/s41587-022-01418-8. doi: 
            10.1038/s41587-022-01418-8."
COMMENT     Sequence Label: pIG-648 CTLA4int1-CTLA4-GFP-SV40
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(38..60)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     160..179
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     373..395
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     387..404
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     388..404
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2052..2564
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVE"
     primer_bind     complement(2091..2112)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2699..2720
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2754..2875
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2791..2810)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2845..2864
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3960..3976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3960..3976)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3973..3995)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3984..4000
                     /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(join(4008..4014,4015..4032,4033..4038))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4053..4074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4191..4208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4362..4950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4442..4461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5121..5912,5913..5981))
                     /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     5744..5763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5982..6086)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6154..6172
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
       61 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
      121 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      181 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
      241 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
      301 cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa
      361 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc cacagtgcta
      421 aggatgctca gagggcatct ctgataggca gaggtgaggg ttagggaagg aagctgtagt
      481 ctagctagct agagctgctg gaatagacat gacaatggct gctgccaaac tgttttctct
      541 tctgaggaca gatgtcccgt gcaagtggct tggtggaagg gactagtgtc tctaatatag
      601 ggtgatttat aagcaggaaa gtgtgtccta gaaattcaga ccagagtgat agattggaat
      661 tggatcatgg gggactcatt gaatgttatt tattgtattt gtttttgcga tcagtgttag
      721 taaagtgtca aagggattga gcagatgagt gacatcatgc aacacaagtt ttgagtttca
      781 cttgtcagac tgactggaga ggggcctggt tagttacagg aaggtaattt ggcatgcagc
      841 cactattttt gagttgatgc aagcctctct gtatggagag ctggtctcct ttatcctgtg
      901 ggaaaagaga acaaaggagc atgggagtgt tcaagggaag gagaaataaa gggcagagag
      961 gcagcggtgg tgtcagggga agcccacagg agttaacagc agggttgcct caacctagag
     1021 aggaagcgac ctggtgccct cggctctgtg gcttccttca tctaacaaca tcttccactc
     1081 tacaacaatg ccagggaagg cggaggctgg tacagtgcat caagacacag ctactcctgg
     1141 gtgacagagg ttcagggcca gctcactaag taggcagaag tttttgacat atactttgag
     1201 agataaagca agattctgta cctcaacctt cagaatttcc cctaccactc attatagttc
     1261 cggagctata tagctcctat cattctatca taaccttaga ataccagaga acatatcatc
     1321 tcatctaatt atctcttact atatgtgaaa aaaatgaagg acatggggga agtgtgactt
     1381 gccccaaatc acatatttca tggtagaggg ctgggcttgg ccatgaagga gcatgagttc
     1441 actgagttcc ctttggcttt tccatgctag caatgcacgt ggcccagcct gctgtggtac
     1501 tggccagcag ccgaggcatc gccagctttg tgtgtgagta tgcatctcca ggcaaagcca
     1561 ctgaggtccg ggtgacagtg cttcggcagg ctgacagcca ggtgactgaa gtctgtgcgg
     1621 caacctacat gatggggaat gagttgacct tcctagatga ttccatctgc acgggcacct
     1681 ccagtggaaa tcaagtgaac ctcactatcc aaggactgag ggccatggac acgggactct
     1741 acatctgcaa ggtggagctc atgtacccac cgccatacta cctgggcata ggcaacggaa
     1801 cccagattta tgtaattgat ccagaaccgt gcccagattc tgacttcctc ctctggatcc
     1861 ttgcagcagt tagttcgggg ttgttttttt atagctttct cctcacagct gtttctttga
     1921 gcaaaatgct aaagaaaaga agccctctta caacaggggt ctatgtgaaa atgcccccaa
     1981 cagagccaga atgtgaaaag caatttcagc cttattttat tcccatcaat ggatctggag
     2041 gaactagcgg cagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2101 tggacggcga cgtaaacggc cacaagttca gcgtgcgcgg cgagggcgag ggcgatgcca
     2161 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2221 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2281 tgaagcgcca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2341 tcagcttcaa ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     2401 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2461 ggcacaagct ggagtacaac ttcaacagcc acaacgtcta tatcaccgcc gacaagcaga
     2521 agaacggcat caaggccaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc
     2581 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2641 accactacct gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca
     2701 tggtcctgct ggagttcgtg accgccgccg ggatcacttg acaccgggtc ttcaacttgt
     2761 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2821 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     2881 tctggaagac ctgtttacct cttctgtttg tcatatcagt gttcttcctg ccacaaccat
     2941 cttgaagaat ctatttctca gtaagaaaat atctttatgg agagtagctg gaaaacagtt
     3001 gagagatgga ggggaggctg ggggtgtgga gaggggaagg ggtaagtgat agattcgttg
     3061 aaggggggag aaaaggccgt ggggatgaag ctagaaggca gaagggcttg cctgggcttg
     3121 gccatgaagg agcatgagtt cactgagttc cctttggctt ttccatgcta ccaatgcacg
     3181 tggcccagcc tgctgtggta ctggccagca gccgaggcat cgccagcttt gtgtgtgagt
     3241 atgcatctcc aggcaaagcc actgaggtcc gggtgacagt gcttcggcag gctgacagcc
     3301 aggtgactga agtctgtgcg gcaacctaca tgatggggaa tgagttgacc ttcctagatg
     3361 attccatctg cacgggcacc tccagtggaa atcaagtgaa cctcactatc caaggactga
     3421 gggccatgga cacgggactc tacatctgca aggtggagct catgtaccca ccgccatact
     3481 acctgggcat aggcaacgga acccagattt atgtaattgg tgagcaaagc catttcactg
     3541 agttgacacc tgttgcattg cagtcatcta tgcacaaaaa cagttttgtt ccttaatttc
     3601 aggaggttta cttttaggac tgtggacatt ctctttaaga gttctgtacc acatggtagc
     3661 cttgcttatt gtgggtggca accttaatag cattctgact gtaaaataaa atgatttggg
     3721 gaagttgggg ctctcgctct ggagtgctaa ccatcatgac gtttgatctg tacttttgat
     3781 atgatatgat gctcctgggg aagtagtccc aaatagccaa acctattggt gggctaccca
     3841 tgcaatttag gggtggacct caaggcctgg aagctctaat gtcctttttt caccaatgtt
     3901 ggggagtaga gccctagtct agagtcgacc tgcaggcatg caagcttggc gtaatcatgg
     3961 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4021 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4141 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4201 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4261 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4321 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4381 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4441 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4501 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4561 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4621 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4681 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4741 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4801 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4861 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4921 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4981 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5041 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5101 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5161 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5221 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5281 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5341 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5401 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5461 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5521 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5581 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5641 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5701 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5761 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5821 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5881 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5941 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6001 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6061 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6121 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gc
//
