LOCUS       sequence-367844-        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-721 CTLA4-GFP T124P
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..57)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     157..176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     370..392
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     384..401
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     385..401
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2049..2561
                     /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(2088..2109)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2696..2717
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2751..2872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2788..2807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2842..2861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3957..3973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3957..3973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3970..3992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3981..3997
                     /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(4005..4011,4012..4029,4030..4035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4050..4071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4188..4205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4359..4947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4439..4458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5118..5909,5910..5978))
                     /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     5741..5760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5979..6083)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6151..6169
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
       61 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      121 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      181 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
      241 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
      301 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
      361 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattccac agtgctaagg
      421 atgctcagag ggcatctctg ataggcagag gtgagggtta gggaaggaag ctgtagtcta
      481 gctagctaga gctgctggaa tagacatgac aatggctgct gccaaactgt tttctcttct
      541 gaggacagat gtcccgtgca agtggcttgg tggaagggac tagtgtctct aatatagggt
      601 gatttataag caggaaagtg tgtcctagaa attcagacca gagtgataga ttggaattgg
      661 atcatggggg actcattgaa tgttatttat tgtatttgtt tttgcgatca gtgttagtaa
      721 agtgtcaaag ggattgagca gatgagtgac atcatgcaac acaagttttg agtttcactt
      781 gtcagactga ctggagaggg gcctggttag ttacaggaag gtaatttggc atgcagccac
      841 tatttttgag ttgatgcaag cctctctgta tggagagctg gtctccttta tcctgtggga
      901 aaagagaaca aaggagcatg ggagtgttca agggaaggag aaataaaggg cagagaggca
      961 gcggtggtgt caggggaagc ccacaggagt taacagcagg gttgcctcaa cctagagagg
     1021 aagcgacctg gtgccctcgg ctctgtggct tccttcatct aacaacatct tccactctac
     1081 aacaatgcca gggaaggcgg aggctggtac agtgcatcaa gacacagcta ctcctgggtg
     1141 acagaggttc agggccagct cactaagtag gcagaagttt ttgacatata ctttgagaga
     1201 taaagcaaga ttctgtacct caaccttcag aatttcccct accactcatt atagttccgg
     1261 agctatatag ctcctatcat tctatcataa ccttagaata ccagagaaca tatcatctca
     1321 tctaattatc tcttactata tgtgaaaaaa atgaaggaca tgggggaagt gtgacttgcc
     1381 ccaaatcaca tatttcatgg tagagggctg ggcttggcca tgaaggagca tgagttcact
     1441 gagttccctt tggcttttcc atgctagcaa tgcacgtggc ccagcctgct gtggtactgg
     1501 ccagcagccg aggcatcgcc agctttgtgt gtgagtatgc atctccaggc aaagccactg
     1561 aggtccgggt gacagtgctt cggcaggctg acagccaggt gactgaagtc tgtgcggcaa
     1621 cctacatgat ggggaatgag ttgaccttcc tagatgattc catctgcacg ggcacctcca
     1681 gtggaaatca agtgaacctc actatccaag gactgagggc catggacccg ggactctaca
     1741 tctgcaaggt ggagctcatg tacccaccgc catactacct gggcataggc aacggaaccc
     1801 agatttatgt aattgatcca gaaccgtgcc cagattctga cttcctcctc tggatccttg
     1861 cagcagttag ttcggggttg tttttttata gctttctcct cacagctgtt tctttgagca
     1921 aaatgctaaa gaaaagaagc cctcttacaa caggggtcta tgtgaaaatg cccccaacag
     1981 agccagaatg tgaaaagcaa tttcagcctt attttattcc catcaatgga tctggaggaa
     2041 ctagcggcag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2101 acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc gatgccacca
     2161 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2221 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2281 agcgccacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatca
     2341 gcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2401 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2461 acaagctgga gtacaacttc aacagccaca acgtctatat caccgccgac aagcagaaga
     2521 acggcatcaa ggccaacttc aagatccgcc acaacgtgga ggacggcagc gtgcagctcg
     2581 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2641 actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc gatcacatgg
     2701 tcctgctgga gttcgtgacc gccgccggga tcacttgaca ccgggtcttc aacttgttta
     2761 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2821 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2881 ggaagacctg tttacctctt ctgtttgtca tatcagtgtt cttcctgcca caaccatctt
     2941 gaagaatcta tttctcagta agaaaatatc tttatggaga gtagctggaa aacagttgag
     3001 agatggaggg gaggctgggg gtgtggagag gggaaggggt aagtgataga ttcgttgaag
     3061 gggggagaaa aggccgtggg gatgaagcta gaaggcagaa gggcttgcct gggcttggcc
     3121 atgaaggagc atgagttcac tgagttccct ttggcttttc catgctacca atgcacgtgg
     3181 cccagcctgc tgtggtactg gccagcagcc gaggcatcgc cagctttgtg tgtgagtatg
     3241 catctccagg caaagccact gaggtccggg tgacagtgct tcggcaggct gacagccagg
     3301 tgactgaagt ctgtgcggca acctacatga tggggaatga gttgaccttc ctagatgatt
     3361 ccatctgcac gggcacctcc agtggaaatc aagtgaacct cactatccaa ggactgaggg
     3421 ccatggacac gggactctac atctgcaagg tggagctcat gtacccaccg ccatactacc
     3481 tgggcatagg caacggaacc cagatttatg taattggtga gcaaagccat ttcactgagt
     3541 tgacacctgt tgcattgcag tcatctatgc acaaaaacag ttttgttcct taatttcagg
     3601 aggtttactt ttaggactgt ggacattctc tttaagagtt ctgtaccaca tggtagcctt
     3661 gcttattgtg ggtggcaacc ttaatagcat tctgactgta aaataaaatg atttggggaa
     3721 gttggggctc tcgctctgga gtgctaacca tcatgacgtt tgatctgtac ttttgatatg
     3781 atatgatgct cctggggaag tagtcccaaa tagccaaacc tattggtggg ctacccatgc
     3841 aatttagggg tggacctcaa ggcctggaag ctctaatgtc cttttttcac caatgttggg
     3901 gagtagagcc ctagtctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca
     3961 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4021 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4081 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4201 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4261 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4321 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4381 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4441 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc taccctgccg
     4501 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4561 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4621 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4681 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4741 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4801 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4861 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4921 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4981 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5041 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5101 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5161 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5221 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5281 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5341 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5401 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5461 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5521 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5581 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5641 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5701 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5761 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5821 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5881 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5941 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6001 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6061 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6121 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ct
//
