LOCUS       sequence-367840-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CTLA4 gene knockin with mutation.
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-720 CTLA4-GFP R75W
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..63)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     163..182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     376..398
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     390..407
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     391..407
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2055..2567
                     /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(2094..2115)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2702..2723
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2757..2878
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2794..2813)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2848..2867
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3963..3979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3963..3979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3976..3998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3987..4003
                     /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(4011..4017,4018..4035,4036..4041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4056..4077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4194..4211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4365..4953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4445..4464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5124..5915,5916..5984))
                     /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     5747..5766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5985..6089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(6157..6172,1..3)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
       61 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
      121 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
      181 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
      241 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
      301 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
      361 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttccacagtg
      421 ctaaggatgc tcagagggca tctctgatag gcagaggtga gggttaggga aggaagctgt
      481 agtctagcta gctagagctg ctggaataga catgacaatg gctgctgcca aactgttttc
      541 tcttctgagg acagatgtcc cgtgcaagtg gcttggtgga agggactagt gtctctaata
      601 tagggtgatt tataagcagg aaagtgtgtc ctagaaattc agaccagagt gatagattgg
      661 aattggatca tgggggactc attgaatgtt atttattgta tttgtttttg cgatcagtgt
      721 tagtaaagtg tcaaagggat tgagcagatg agtgacatca tgcaacacaa gttttgagtt
      781 tcacttgtca gactgactgg agaggggcct ggttagttac aggaaggtaa tttggcatgc
      841 agccactatt tttgagttga tgcaagcctc tctgtatgga gagctggtct cctttatcct
      901 gtgggaaaag agaacaaagg agcatgggag tgttcaaggg aaggagaaat aaagggcaga
      961 gaggcagcgg tggtgtcagg ggaagcccac aggagttaac agcagggttg cctcaaccta
     1021 gagaggaagc gacctggtgc cctcggctct gtggcttcct tcatctaaca acatcttcca
     1081 ctctacaaca atgccaggga aggcggaggc tggtacagtg catcaagaca cagctactcc
     1141 tgggtgacag aggttcaggg ccagctcact aagtaggcag aagtttttga catatacttt
     1201 gagagataaa gcaagattct gtacctcaac cttcagaatt tcccctacca ctcattatag
     1261 ttccggagct atatagctcc tatcattcta tcataacctt agaataccag agaacatatc
     1321 atctcatcta attatctctt actatatgtg aaaaaaatga aggacatggg ggaagtgtga
     1381 cttgccccaa atcacatatt tcatggtaga gggctgggct tggccatgaa ggagcatgag
     1441 ttcactgagt tccctttggc ttttccatgc tagcaatgca cgtggcccag cctgctgtgg
     1501 tactggccag cagccgaggc atcgccagct ttgtgtgtga gtatgcatct ccaggcaaag
     1561 ccactgaggt ccgggtgaca gtgctttggc aggctgacag ccaggtgact gaagtctgtg
     1621 cggcaaccta catgatgggg aatgagttga ccttcctaga tgattccatc tgcacgggca
     1681 cctccagtgg aaatcaagtg aacctcacta tccaaggact gagggccatg gacacgggac
     1741 tctacatctg caaggtggag ctcatgtacc caccgccata ctacctgggc ataggcaacg
     1801 gaacccagat ttatgtaatt gatccagaac cgtgcccaga ttctgacttc ctcctctgga
     1861 tccttgcagc agttagttcg gggttgtttt tttatagctt tctcctcaca gctgtttctt
     1921 tgagcaaaat gctaaagaaa agaagccctc ttacaacagg ggtctatgtg aaaatgcccc
     1981 caacagagcc agaatgtgaa aagcaatttc agccttattt tattcccatc aatggatctg
     2041 gaggaactag cggcagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2101 agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg
     2161 ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2221 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     2281 acatgaagcg ccacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2341 ccatcagctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg
     2401 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2461 tggggcacaa gctggagtac aacttcaaca gccacaacgt ctatatcacc gccgacaagc
     2521 agaagaacgg catcaaggcc aacttcaaga tccgccacaa cgtggaggac ggcagcgtgc
     2581 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2641 acaaccacta cctgagcacc cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc
     2701 acatggtcct gctggagttc gtgaccgccg ccgggatcac ttgacaccgg gtcttcaact
     2761 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2821 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     2881 atgtctggaa gacctgttta cctcttctgt ttgtcatatc agtgttcttc ctgccacaac
     2941 catcttgaag aatctatttc tcagtaagaa aatatcttta tggagagtag ctggaaaaca
     3001 gttgagagat ggaggggagg ctgggggtgt ggagagggga aggggtaagt gatagattcg
     3061 ttgaaggggg gagaaaaggc cgtggggatg aagctagaag gcagaagggc ttgcctgggc
     3121 ttggccatga aggagcatga gttcactgag ttccctttgg cttttccatg ctaccaatgc
     3181 acgtggccca gcctgctgtg gtactggcca gcagccgagg catcgccagc tttgtgtgtg
     3241 agtatgcatc tccaggcaaa gccactgagg tccgggtgac agtgcttcgg caggctgaca
     3301 gccaggtgac tgaagtctgt gcggcaacct acatgatggg gaatgagttg accttcctag
     3361 atgattccat ctgcacgggc acctccagtg gaaatcaagt gaacctcact atccaaggac
     3421 tgagggccat ggacacggga ctctacatct gcaaggtgga gctcatgtac ccaccgccat
     3481 actacctggg cataggcaac ggaacccaga tttatgtaat tggtgagcaa agccatttca
     3541 ctgagttgac acctgttgca ttgcagtcat ctatgcacaa aaacagtttt gttccttaat
     3601 ttcaggaggt ttacttttag gactgtggac attctcttta agagttctgt accacatggt
     3661 agccttgctt attgtgggtg gcaaccttaa tagcattctg actgtaaaat aaaatgattt
     3721 ggggaagttg gggctctcgc tctggagtgc taaccatcat gacgtttgat ctgtactttt
     3781 gatatgatat gatgctcctg gggaagtagt cccaaatagc caaacctatt ggtgggctac
     3841 ccatgcaatt taggggtgga cctcaaggcc tggaagctct aatgtccttt tttcaccaat
     3901 gttggggagt agagccctag tctagagtcg acctgcaggc atgcaagctt ggcgtaatca
     3961 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     4021 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4081 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4141 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4201 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4261 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4321 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4381 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4441 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4501 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4561 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4621 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4681 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4741 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4801 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4861 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4921 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4981 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5041 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5101 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5161 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5221 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5281 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5341 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5401 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5461 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5521 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5581 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5641 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5701 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5761 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5821 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5881 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5941 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6001 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6061 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6121 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac ga
//
