LOCUS       sequence-210635-        3784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DNA sequence source for amplifying an HDR template to replace 
            endogenous human TCR-alpha with 1G4 NYESO TCR-alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3784)
  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 3784)
  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 into TRAC HDRT Source (pTR 223)
FEATURES             Location/Qualifiers
     source          1..3784
                     /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             734..790
                     /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(1563..1579)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1563..1579)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1576..1598)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1587..1603
                     /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(1611..1617,1618..1635,1636..1641))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1656..1677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1794..1811)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1965..2553)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2045..2064)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2724..3515,3516..3584))
                     /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     3347..3366
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3585..3689)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3757..3775
                     /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 ttcaggtttc cttgagtggc aggccaggcc tggccgtgaa cgttcactga aatcatggcc
      481 tcttggccaa gattgatagc ttgtgcctgt ccctgagtcc cagtccatca cgagcagctg
      541 gtttctaaga tgctatttcc cgtataaagc atgagaccgt gacttgccag ccccacagag
      601 ccccgccctt gtccatcact ggcatctgga ctccagcctg ggttggggca aagagggaaa
      661 tgagatcatg tcctaaccct gatcctcttg tcccacagat atccagaacc ctgaccctgc
      721 ctccggatcc ggagccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa
      781 ccccggcccc atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt
      841 gagcagcaaa caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa
      901 cttggttctc aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca
      961 ggaccctggg aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac
     1021 aagtggaaga cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc
     1081 agcttctcag cctggtgact cagccaccta cctctgtgct gtgaggcccc tgtacggagg
     1141 aagctacata cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa
     1201 ccctgaccct gcggtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct
     1261 attcaccgat tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat
     1321 cacagacaaa actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc
     1381 ctggagcaac aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga
     1441 agacaccttc ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct
     1501 tgcttcagga atggccatcc tctagagtcg acctgcaggc atgcaagctt ggcgtaatca
     1561 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     1621 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     1681 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     1741 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     1801 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1861 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1921 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1981 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2041 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2101 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2161 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2221 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2281 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2341 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2401 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2461 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2521 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2581 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2641 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2701 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2761 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2821 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2881 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2941 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3001 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3061 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3121 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3181 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3241 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3301 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3361 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3421 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3481 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3541 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3601 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3661 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     3721 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     3781 cgtc
//
