LOCUS       sequence-215612-        4712 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 4712)
  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 4712)
  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..4712
                     /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             1141..1653
                     /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(1180..1201)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1788..1809
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2491..2507)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2491..2507)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2504..2526)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2515..2531
                     /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(2539..2545,2546..2563,2564..2569))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2584..2605
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(2893..3481)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2973..2992)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3652..4443,4444..4512))
                     /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     4275..4294
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4513..4617)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4685..4703
                     /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 aggattcaat cttcttcttc tttttttttt
      541 aggattcaat cttctaataa tcccgggagg gggcgcaaga gcaggattct tgtttctatt
      601 ttacgaatga ggaaactgag gttcagaaag cctaagtggc atgcctcttc agtaatcggc
      661 caatcgtcgc agccctaact tcacctcacc tcccagtgca gcactagagt ccctcagggg
      721 tggccgtccc tggtcagtcc agctccagca gacggggtgg gtaggacgct ccctatcagg
      781 cagcacttcc gcctcccggg gcccgcgcag ctcacctccc tcacctcccg ccctacccca
      841 gtcacgagtt gttttagggg gaccgcccct ccacttgctg attgggtagc tcctgaacca
      901 ttgttgtcct ctgattggtt gttccctttt cggctctgca acaccgccta gaccgaccgg
      961 atacacgggt agggcttccg ctttacccgt ctccctcctg gcgcttgtcc tcctctccca
     1021 gtcggcacca cagcggtggc tgccgggcgt ggtgtcggtg ggtcggttgg tttttgtctc
     1081 accgttggtg tccgtgccgt tcagttgccc gccatggctg gatcgggtgg gactagtggc
     1141 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1201 gtaaacggcc acaagttcag cgtgcgcggc gagggcgagg gcgatgccac caacggcaag
     1261 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1321 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcgccac
     1381 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cagcttcaag
     1441 gacgacggca cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1501 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1561 gagtacaact tcaacagcca caacgtctat atcaccgccg acaagcagaa gaacggcatc
     1621 aaggccaact tcaagatccg ccacaacgtg gaggacggca gcgtgcagct cgccgaccac
     1681 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1741 agcacccagt ccgtgctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1801 gagttcgtga ccgccgccgg gatcactgga accggtgctg gaagtggtga gctggatccg
     1861 ttcggcgccc ctgccggcgc ccctggcggt cccgcgctgg ggaacggagt ggccggcgcc
     1921 ggcgaagaag acccggctgc ggccttcttg gcgcagcaag agagcgagat tgcgggcatc
     1981 gagaacgacg aggccttcgc catcctggac ggcggcgccc ccgggcccca gccgcacggc
     2041 gagccgccgg ggggtccggg tgagagtgcg ggcgcgtttg gggcgagagg acttgtctgg
     2101 aaactcggtc cacagtgggt ccgagagctt ctgtgtgact cgtgctcctt gctgaattag
     2161 gaggttaggg agcagtgcaa acaggaaacg agaccctggc ccggtctttc agaaacctag
     2221 gctcgagaag cctgttcggt tctcagcatg tttgagtgct tctgggcgcg ggcggagcga
     2281 gaaagcaagt gtagggtggc aggctccgga gccggaagaa gcccgttcaa ttcagcaact
     2341 tttcattaag catttgctgt gcctttagtc cgggcttcct actatcgtca gagacttccc
     2401 tgctcacttg tttgccctca ggctcgagtc ctcgctcact gactctcctc tagagtcgac
     2461 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     2521 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2581 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2641 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2701 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2761 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2821 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2881 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2941 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3001 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3061 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3121 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3181 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3241 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3301 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3361 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3421 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3481 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3541 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3601 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3661 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3721 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3781 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3841 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3901 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3961 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4021 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4081 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4141 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4201 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4261 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4321 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4381 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4441 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4501 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4561 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4621 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     4681 taaaaatagg cgtatcacga ggccctttcg tc
//
