LOCUS       sequence-215608-        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     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..855
                     /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(1628..1644)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1628..1644)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1641..1663)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1652..1668
                     /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(1676..1682,1683..1700,1701..1706))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1721..1742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1859..1876)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2030..2618)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2110..2129)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2789..3580,3581..3649))
                     /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     3412..3431
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3650..3754)
                     /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 gatccggagc caccaacttc agcctgctga agcaggccgg cgacgtggag
      841 gagaaccccg gccccatgga gaccctcttg ggcctgctta tcctttggct gcagctgcaa
      901 tgggtgagca gcaaacagga ggtgacgcag attcctgcag ctctgagtgt cccagaagga
      961 gaaaacttgg ttctcaactg cagtttcact gatagcgcta tttacaacct ccagtggttt
     1021 aggcaggacc ctgggaaagg tctcacatct ctgttgctta ttcagtcaag tcagagagag
     1081 caaacaagtg gaagacttaa tgcctcgctg gataaatcat caggacgtag tactttatac
     1141 attgcagctt ctcagcctgg tgactcagcc acctacctct gtgctgtgag gcccctgtac
     1201 ggaggaagct acatacctac atttggaaga ggaaccagcc ttattgttca tccgtatatc
     1261 cagaaccctg accctgcggt gtaccagctg agagactcta aatccagtga caagtctgtc
     1321 tgcctattca ccgattttga ttctcaaaca aatgtgtcac aaagtaagga ttctgatgtg
     1381 tatatcacag acaaaactgt gctagacatg aggtctatgg acttcaagag caacagtgct
     1441 gtggcctgga gcaacaaatc tgactttgca tgtgcaaacg ccttcaacaa cagcattatt
     1501 ccagaagaca ccttcttccc cagcccaggt aagggcagct ttggtgcctt cgcaggctgt
     1561 ttccttgctt caggaatggc catcctctag agtcgacctg caggcatgca agcttggcgt
     1621 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     1681 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     1741 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     1801 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1861 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1921 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     1981 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2041 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2101 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2161 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2221 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2281 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2341 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2401 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2461 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2521 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2581 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2641 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2701 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2761 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2821 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2881 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     2941 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3001 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3061 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3121 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3181 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3241 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3301 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3361 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3421 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3481 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3541 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3601 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3661 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3721 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     3781 acgt
//
