LOCUS       sequence-215596-        4795 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-alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4795)
  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 4795)
  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 beta/alpha into TRAC HDRT Source (pTR 169)
FEATURES             Location/Qualifiers
     source          1..4795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     38..56
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(94..116)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     216..235
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     429..451
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     443..460
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     444..460
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             799..852
                     /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             1810..1866
                     /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(2639..2655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2639..2655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2652..2674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2663..2679
                     /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(2687..2693,2694..2711,2712..2717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2732..2753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2870..2887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3041..3629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3121..3140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3800..4591,4592..4660))
                     /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     4423..4442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4661..4765)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
       61 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
      121 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
      181 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
      241 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      301 gcgccattca ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
      361 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
      421 agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc tcggtacccg
      481 gggatttcag gtttccttga gtggcaggcc aggcctggcc gtgaacgttc actgaaatca
      541 tggcctcttg gccaagattg atagcttgtg cctgtccctg agtcccagtc catcacgagc
      601 agctggtttc taagatgcta tttcccgtat aaagcatgag accgtgactt gccagcccca
      661 cagagccccg cccttgtcca tcactggcat ctggactcca gcctgggttg gggcaaagag
      721 ggaaatgaga tcatgtccta accctgatcc tcttgtccca cagatatcca gaaccctgac
      781 cctgcctccg gatccggaga gggcagggga tctctcctta cttgtggcga cgtggaggag
      841 aaccccggcc ccatgagcat cggcctcctg tgctgtgcag ccttgtctct cctgtgggca
      901 ggtccagtga atgctggtgt cactcagacc ccaaaattcc aggtcctgaa gacaggacag
      961 agcatgacac tgcagtgtgc ccaggatatg aaccatgaat acatgtcctg gtatcgacaa
     1021 gacccaggca tggggctgag gctgattcat tactcagttg gtgctggtat cactgaccaa
     1081 ggagaagtcc ccaatggcta caatgtctcc agatcaacca cagaggattt cccgctcagg
     1141 ctgctgtcgg ctgctccctc ccagacatct gtgtacttct gtgccagcag ttacgtcggg
     1201 aacaccgggg agctgttttt tggagaaggc tctaggctga ccgtactgga ggacctgaaa
     1261 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc
     1321 caaaaggcca cactggtatg cctggccaca ggcttctacc ccgaccacgt ggagctgagc
     1381 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag
     1441 gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc
     1501 accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg
     1561 gagaatgacg agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag
     1621 gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct
     1681 gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt
     1741 gccctcgtgc tgatggctat ggtcaagaga aaggattcca gaggccgggc caagcggtcc
     1801 ggatccggag ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc
     1861 ggccccatgg agaccctctt gggcctgctt atcctttggc tgcagctgca atgggtgagc
     1921 agcaaacagg aggtgacgca gattcctgca gctctgagtg tcccagaagg agaaaacttg
     1981 gttctcaact gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac
     2041 cctgggaaag gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt
     2101 ggaagactta atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct
     2161 tctcagcctg gtgactcagc cacctacctc tgtgctgtga ggcccctgta cggaggaagc
     2221 tacataccta catttggaag aggaaccagc cttattgttc atccgtatat ccagaaccct
     2281 gaccctgcgg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc
     2341 accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca
     2401 gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg
     2461 agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac
     2521 accttcttcc ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct
     2581 tcaggaatgg ccatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt
     2641 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     2701 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     2761 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     2821 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     2881 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     2941 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3001 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3061 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3121 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3181 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3241 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3301 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3361 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3421 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     3481 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     3541 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     3601 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     3661 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     3721 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     3781 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     3841 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     3901 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     3961 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4021 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4081 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4141 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4201 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4261 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4321 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4381 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4441 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     4501 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     4561 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     4621 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     4681 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4741 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgt
//
