LOCUS       sequence-210644-        4713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to tag endogenous
            human CLTA gene with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4713)
  AUTHORS   Roth TL, Puig-Saus C, Yu R, Shifrut E, Carnevale J, Li PJ, Hiatt J, 
            Saco J, Krystofinski P, Li H, Tobin V, Nguyen DN, Lee MR, Putnam AL,
            Ferris AL, Chen JW, Schickel JN, Pellerin L, Carmody D, 
            Alkorta-Aranburu G, Del Gaudio D, Matsumoto H, Morell M, Mao Y, Cho 
            M, Quadros RM, Gurumurthy CB, Smith B, Haugwitz M, Hughes SH, 
            Weissman JS, Schumann K, Esensten JH, May AP, Ashworth A, Kupfer GM,
            Greeley SAW, Bacchetta R, Meffre E, Roncarolo MG, Romberg N, Herold 
            KC, Ribas A, Leonetti MD, Marson A
  TITLE     Reprogramming human T cell function and specificity with non-viral 
            genome targeting.
  JOURNAL   Nature. 2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 
            10.1038/s41586-018-0326-5.
  PUBMED    29995861
REFERENCE   2  (bases 1 to 4713)
  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: "Nature. 
            2018 Jul 11. pii: 10.1038/s41586-018-0326-5. doi: 
            10.1038/s41586-018-0326-5."
COMMENT     Sequence Label: CLTA-GFP HDRT Source (pTR 153)
FEATURES             Location/Qualifiers
     source          1..4713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     475..494
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        475..493
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1142..1654
                     /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(1181..1202)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1789..1810
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2492..2508)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2492..2508)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2505..2527)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2516..2532
                     /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(2540..2546,2547..2564,2565..2570))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2585..2606
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2894..3482)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2974..2993)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3653..4444,4445..4513))
                     /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     4276..4295
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4514..4618)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4686..4704
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggag
      421 tactgagagt gcaccaggat ccgggaacct cttctgtaac tccttagcag ctgataatac
      481 gactcactat agggaccctc aaatccataa aaggattcaa tcttcttctt cttttttttt
      541 aggattcaat cttctaataa tcccgggagg gggcgcaaga gcaggattct tgtttctatt
      601 ttacgaatga ggaaactgag gttcagaaag cctaagtggc atgcctcttc agtaatcggc
      661 caatcgtcgc tgccctaact tcacctcacc tcccagtgca gcactagagt cccctcaggg
      721 gtggccgtcc ctggtcagtc cagctccagc agacggggtg ggtaggacgc tccctatcag
      781 gcagcacttc cgcctcccgg ggcccgcgca gctcacctcc ctcacctccc gccctacccc
      841 agtcacgagt tgttttaggg ggaccgcccc tccacttgct gattgggtag ctcctgaacc
      901 attgttgtcc tctgattggt tgttcccttt tcggctctgc aacaccgcct agaccgaccg
      961 gatacacggg tagggcttcc gctttacccg tctccctcct ggcgcttgtc ctcctctccc
     1021 agtcggcacc acagcggtgg ctgccgggcg tggtgtcggt gggtcggttg gtttttgtct
     1081 caccgttggt gtccgtgccg ttcagttgcc cgccatggct ggatcgggtg ggactagtgg
     1141 cagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1201 cgtaaacggc cacaagttca gcgtgcgcgg cgagggcgag ggcgatgcca ccaacggcaa
     1261 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1321 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcgcca
     1381 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcagcttcaa
     1441 ggacgacggc acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1501 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1561 ggagtacaac ttcaacagcc acaacgtcta tatcaccgcc gacaagcaga agaacggcat
     1621 caaggccaac ttcaagatcc gccacaacgt ggaggacggc agcgtgcagc tcgccgacca
     1681 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1741 gagcacccag tccgtgctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1801 ggagttcgtg accgccgccg ggatcactgg aaccggtgct ggaagtggtg agctggatcc
     1861 gttcggcgcc cctgccggcg cccctggcgg tcccgcgctg gggaacggag tggccggcgc
     1921 cggcgaagaa gacccggctg cggccttctt ggcgcagcaa gagagcgaga ttgcgggcat
     1981 cgagaacgac gaggccttcg ccatcctgga cggcggcgcc cccgggcccc agccgcacgg
     2041 cgagccgccg gggggtccgg gtgagagtgc gggcgcgttt ggggcgagag gacttgtctg
     2101 gaaactcggt ccacagtggg tccgagagct tctgtgtgac tcgtgctcct tgctgaatta
     2161 ggaggttagg gagcagtgca aacaggaaac gagaccctgg cccggtcttt cagaaaccta
     2221 ggctcgagaa gcctgttcgg ttctcagcat gtttgagtgc ttctgggcgc gggcggagcg
     2281 agaaagcaag tgtagggtgg caggctccgg agccggaaga agcccgttca attcagcaac
     2341 ttttcattaa gcatttgctg tgcctttagt ccgggcttcc tactatcgtc agagacttcc
     2401 ctgctcactt gtttgccctc aggctcgagt cctcgctcac tgactctcct ctagagtcga
     2461 cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2521 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2581 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2641 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2701 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2761 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2821 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2881 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2941 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3001 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3061 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3121 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3181 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3241 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3301 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3361 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3421 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3481 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3541 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3601 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3661 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3721 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3781 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3841 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3901 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3961 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4021 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4081 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4141 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4201 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4261 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4321 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4381 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4441 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4501 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4561 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4621 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4681 ataaaaatag gcgtatcacg aggccctttc gtc
//
