LOCUS       sequence-210650-        4833 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to replace 
            endogenous human TCR with 1G4 NYESO TCR at TCR-beta.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4833)
  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 4833)
  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: NYESO alpha/beta into TRBC1 HDRT Source (pTR 262)
FEATURES             Location/Qualifiers
     source          1..4833
                     /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"
     CDS             826..879
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1726..1782
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2612..2628)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2612..2628)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2625..2647)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2636..2652
                     /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(2660..2666,2667..2684,2685..2690))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2705..2726
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2843..2860)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3014..3602)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3094..3113)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3773..4564,4565..4633))
                     /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     4396..4415
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4634..4738)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4806..4824
                     /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 acccggggat
      421 aatgagagtt tcctacctcg agtttcagga ttacatagcc atgcaccaag caaggctttg
      481 aaaaataaag atacacagat aaattatttg gatagatgat cagacaagcc tcagtaaaaa
      541 cagccaagac aatcaggata taatgtgacc ataggaagct ggggagacag taggcaatgt
      601 gcatccatgg gacagcatag aaaggagggg caaagtggag agagagcaac agacactggg
      661 atggtgaccc caaaacaatg agggcctaga atgacatagt tgtgcttcat tacggcccat
      721 tcccagggct ctctctcaca cacacagagc ccctaccaga accagacagc tctcagagca
      781 accctggctc caacccctct tccctttcca gagtccggat ccggagaggg caggggatct
      841 ctccttactt gtggcgacgt ggaggagaac cccggcccca tggagaccct cttgggcctg
      901 cttatccttt ggctgcagct gcaatgggtg agcagcaaac aggaggtgac gcagattcct
      961 gcagctctga gtgtcccaga aggagaaaac ttggttctca actgcagttt cactgatagc
     1021 gctatttaca acctccagtg gtttaggcag gaccctggga aaggtctcac atctctgttg
     1081 cttattcagt caagtcagag agagcaaaca agtggaagac ttaatgcctc gctggataaa
     1141 tcatcaggac gtagtacttt atacattgca gcttctcagc ctggtgactc agccacctac
     1201 ctctgtgctg tgaggcccct gtacggagga agctacatac ctacatttgg aagaggaacc
     1261 agccttattg ttcatccgta tatccagaac cctgaccctg ccgtgtacca gctgagagac
     1321 tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca aacaaatgtg
     1381 tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga catgaggtct
     1441 atggacttca agagcaacag tgctgtggcc tggagcaaca aatctgactt tgcatgtgca
     1501 aacgccttca acaacagcat tattccagaa gacaccttct tccccagccc agaaagttcc
     1561 tgtgatgtca agctggtcga gaaaagcttt gaaacagata cgaacctaaa ctttcaaaac
     1621 ctgtcagtga ttgggttccg aatcctcctc ctgaaagtgg ccgggtttaa tctgctcatg
     1681 acgctgcggc tgtggtccag ccgggccaag cggtccggat ccggagccac caacttcagc
     1741 ctgctgaagc aggccggcga cgtggaggag aaccccggcc ccatgagcat cggcctcctg
     1801 tgctgtgcag ccttgtctct cctgtgggca ggtccagtga atgctggtgt cactcagacc
     1861 ccaaaattcc aggtcctgaa gacaggacag agcatgacac tgcagtgtgc ccaggatatg
     1921 aaccatgaat acatgtcctg gtatcgacaa gacccaggca tggggctgag gctgattcat
     1981 tactcagttg gtgctggtat cactgaccaa ggagaagtcc ccaatggcta caatgtctcc
     2041 agatcaacca cagaggattt cccgctcagg ctgctgtcgg ctgctccctc ccagacatct
     2101 gtgtacttct gtgccagcag ttacgtcggg aacaccgggg agctgttttt tggagaaggc
     2161 tctaggctga ccgtactgga ggacctgaac aaagtgttcc cacccgaggt cgctgtgttt
     2221 gagccatcag aagcagagat ctcccacacc caaaaggcca cactggtgtg cctggccaca
     2281 ggcttcttcc ctgaccacgt ggagctgagc tggtgggtga atgggaagga ggtgcacagt
     2341 ggggtcagca cggacccgca gcccctcaag gagcagcccg ccctcaatga ctccagatac
     2401 tgcctgagca gccgcctgag ggtctcggcc accttctggc agaacccccg caaccacttc
     2461 cgctgtcaag tccagttcta cgggctctcg gagaatgacg agtggaccca ggatagggcc
     2521 aaacccgtca cccagatcgt cagcgccgag gcctggggta gagcagtcct ctagagtcga
     2581 cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2641 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2701 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2761 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2821 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2881 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2941 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3001 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3061 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3121 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3181 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3241 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3301 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3361 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3421 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3481 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3541 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3601 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3661 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3721 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3781 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3841 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3901 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3961 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4021 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4081 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4141 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4201 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4261 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4321 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4381 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4441 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4501 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4561 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4621 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4681 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4741 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4801 ataaaaatag gcgtatcacg aggccctttc gtc
//
