LOCUS       sequence-412309-        7243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid can be used to generate HDR templates for non-viral 
            knockin into the TRAC locus of human T cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7243)
  AUTHORS   Blaeschke F, Chen YY, Apathy R, Daniel B, Chen AY, Chen PA, Sandor 
            K, Zhang W, Li Z, Mowery CT, Yamamoto TN, Nyberg WA, To A, Yu R, 
            Bueno R, Kim MC, Schmidt R, Goodman DB, Feuchtinger T, Eyquem J, 
            Jimmie Ye C, Carnevale J, Satpathy AT, Shifrut E, Roth TL, Marson A
  TITLE     Modular pooled discovery of synthetic knockin sequences to program 
            durable cell therapies.
  JOURNAL   Cell. 2023 Sep 14;186(19):4216-4234.e33. doi: 
            10.1016/j.cell.2023.08.013.
  PUBMED    37714135
REFERENCE   2  (bases 1 to 7243)
  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"; doi: 
            "10.1016/j.cell.2023.08"; journalName: "Cell"; date: "2023-09-14- 
            14"; volume: "186"; issue: "19"; pages: "4216-4234.e33"
COMMENT     Sequence Label: pIG-834_NY-ESO-1_TCR_FAS/IL4R
FEATURES             Location/Qualifiers
     source          1..7243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1260..1282)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1382..1401
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1595..1617
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1609..1626
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1610..1626
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1992..2051
                     /codon_start=1
                     /product="2A peptide from equine rhinitis A virus 
                     polyprotein"
                     /label=E2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="QCTNYALLKLAGDVESNPGP"
     CDS             4413..4466
                     /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             5424..5480
                     /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(6253..6269)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6253..6269)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6266..6288)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6277..6293
                     /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(6301..6307,6308..6325,6326..6331))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6346..6367
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6484..6501)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6655..7243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6735..6754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     1321 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     1381 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     1441 tgcgtaagga gaaaataccg catcaggcgc cattcaccat tcaggctgcg caactgttgg
     1501 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1561 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1621 gccagtgaat tcgagctcgg tacccgggga tttcaggttt ccttgagtgg caggccaggc
     1681 ctggccgtga acgttcactg aaatcatggc ctcttggcca agattgatag cttgtgcctg
     1741 tccctgagtc ccagtccatc acgagcagct ggtttctaag atgctatttc ccgtataaag
     1801 catgagcacg tgcattcgca cgcccacaga gccccgccct tgtccatcac tggcatctgg
     1861 actccagcct gggttggggc aaagagggaa atgagatcat gtcctaaccc tgatcctctt
     1921 gtcccacaga tatccagaac cctgaccctg ccggatcggg tgggactagt ggcggcgggg
     1981 ggagcggcgg acagtgcacc aactatgcgc tgctgaaact ggcgggcgat gtggaaagca
     2041 acccgggccc gatgctgggc atctggaccc tcctacctct ggttcttacg tctgttgcta
     2101 gattatcgtc caaaagtgtt aatgcccaag tgactgacat caactccaag ggattggaat
     2161 tgaggaagac tgttactaca gttgagactc agaacttgga aggcctgcat catgatggcc
     2221 aattctgcca taagccctgt cctccaggtg aaaggaaagc tagggactgc acagtcaatg
     2281 gggatgaacc agactgcgtg ccctgccaag aagggaagga gtacacagac aaagcccatt
     2341 tttcttccaa atgcagaaga tgtagattgt gtgatgaagg acatggctta gaagtggaaa
     2401 taaactgcac ccggacccag aataccaagt gcagatgtaa accaaacttt ttttgtaact
     2461 ctactgtatg tgaacactgt gacccttgca ccaaatgtga acatggaatc atcaaggaat
     2521 gcacactcac cagcaacacc aagtgcaaag aggaaggatc cagatctaac ttggggtggc
     2581 tttgtcttct tcttttgcca attccactaa ttgtttgggt gaagagaaag gaagtacaga
     2641 aaaagattaa gaaagaatgg tgggatcaga ttcccaaccc agcccgcagc cgcctcgtgg
     2701 ctataataat ccaggatgct caggggtcac agtgggagaa gcggtcccga ggccaggaac
     2761 cagccaagtg cccacactgg aagaattgtc ttaccaagct cttgccctgt tttctggagc
     2821 acaacatgaa aagggatgaa gatcctcaca aggctgccaa agagatgcct ttccagggct
     2881 ctggaaaatc agcatggtgc ccagtggaga tcagcaagac agtcctctgg ccagagagca
     2941 tcagcgtggt gcgatgtgtg gagttgtttg aggccccggt ggagtgtgag gaggaggagg
     3001 aggtagagga agaaaaaggg agcttctgtg catcgcctga gagcagcagg gatgacttcc
     3061 aggagggaag ggagggcatt gtggcccggc taacagagag cctgttcctg gacctgctcg
     3121 gagaggagaa tgggggcttt tgccagcagg acatggggga gtcatgcctt cttccacctt
     3181 cgggaagtac gagtgctcac atgccctggg atgagttccc aagtgcaggg cccaaggagg
     3241 cacctccctg gggcaaggag cagcctctcc acctggagcc aagtcctcct gccagcccga
     3301 cccagagtcc agacaacctg acttgcacag agacgcccct cgtcatcgca ggcaaccctg
     3361 cttaccgcag cttcagcaac tccctgagcc agtcaccgtg tcccagagag ctgggtccag
     3421 acccactgct ggccagacac ctggaggaag tagaacccga gatgccctgt gtcccccagc
     3481 tctctgagcc aaccactgtg ccccaacctg agccagaaac ctgggagcag atcctccgcc
     3541 gaaatgtcct ccagcatggg gcagctgcag cccccgtctc ggcccccacc agtggctatc
     3601 aggagtttgt acatgcggtg gagcagggtg gcacccaggc cagtgcggtg gtgggcttgg
     3661 gtcccccagg agaggctggt tacaaggcct tctcaagcct gcttgccagc agtgctgtgt
     3721 ccccagagaa atgtgggttt ggggctagca gtggggaaga ggggtataag cctttccaag
     3781 acctcattcc tggctgccct ggggaccctg ccccagtccc tgtccccttg ttcacctttg
     3841 gactggacag ggagccacct cgcagtccgc agagctcaca tctcccaagc agctccccag
     3901 agcacctggg tctggagccg ggggaaaagg tagaggacat gccaaagccc ccacttcccc
     3961 aggagcaggc cacagacccc cttgtggaca gcctgggcag tggcattgtc tactcagccc
     4021 ttacctgcca cctgtgcggc cacctgaaac agtgtcatgg ccaggaggat ggtggccaga
     4081 cccctgtcat ggccagtcct tgctgtggct gctgctgtgg agacaggtcc tcgcccccta
     4141 caacccccct gagggcccca gacccctctc caggtggggt tccactggag gccagtctgt
     4201 gtccggcctc cctggcaccc tcgggcatct cagagaagag taaatcctca tcatccttcc
     4261 atcctgcccc tggcaatgct cagagctcaa gccagacccc caaaatcgtg aactttgtct
     4321 ccgtgggacc cacatacatg agggtctcta gggctaagag aggttcagga tctggggggg
     4381 ggtctggcgg aggaaccggt gctggaagtg gtgagggcag gggatctctc cttacttgtg
     4441 gcgacgtgga ggagaacccc ggccccatga gcatcggcct cctgtgctgt gcagccttgt
     4501 ctctcctgtg ggcaggtcca gtgaatgctg gtgtcactca gaccccaaaa ttccaggtcc
     4561 tgaagacagg acagagcatg acactgcagt gtgcccagga tatgaaccat gaatacatgt
     4621 cctggtatcg acaagaccca ggcatggggc tgaggctgat tcattactca gttggtgctg
     4681 gtatcactga ccaaggagaa gtccccaatg gctacaatgt ctccagatca accacagagg
     4741 atttcccgct caggctgctg tcggctgctc cctcccagac atctgtgtac ttctgtgcca
     4801 gcagttacgt cgggaacacc ggggagctgt tttttggaga aggctctagg ctgaccgtac
     4861 tggaggacct gaaaaacgtg ttcccacccg aggtcgctgt gtttgagcca tcagaagcag
     4921 agatctccca cacccaaaag gccacactgg tatgcctggc cacaggcttc taccccgacc
     4981 acgtggagct gagctggtgg gtgaatggga aggaggtgca cagtggggtc agcacagacc
     5041 cgcagcccct caaggagcag cccgccctca atgactccag atactgcctg agcagccgcc
     5101 tgagggtctc ggccaccttc tggcagaacc cccgcaacca cttccgctgt caagtccagt
     5161 tctacgggct ctcggagaat gacgagtgga cccaggatag ggccaaaccc gtcacccaga
     5221 tcgtcagcgc cgaggcctgg ggtagagcag actgtggctt cacctccgag tcttaccagc
     5281 aaggggtcct gtctgccacc atcctctatg agatcttgct agggaaggcc accttgtatg
     5341 ccgtgctggt cagtgccctc gtgctgatgg ctatggtcaa gagaaaggat tccagaggcc
     5401 gggccaagcg gtccggatcc ggagccacca acttcagcct gctgaagcag gccggcgacg
     5461 tggaggagaa ccccggcccc atggagaccc tcttgggcct gcttatcctt tggctgcagc
     5521 tgcaatgggt gagcagcaaa caggaggtga cgcagattcc tgcagctctg agtgtcccag
     5581 aaggagaaaa cttggttctc aactgcagtt tcactgatag cgctatttac aacctccagt
     5641 ggtttaggca ggaccctggg aaaggtctca catctctgtt gcttattcag tcaagtcaga
     5701 gagagcaaac aagtggaaga cttaatgcct cgctggataa atcatcagga cgtagtactt
     5761 tatacattgc agcttctcag cctggtgact cagccaccta cctctgtgct gtgaggcccc
     5821 tgtacggagg aagctacata cctacatttg gaagaggaac cagccttatt gttcatccgt
     5881 atatccagaa ccctgaccct gcggtgtacc agctgagaga ctctaaatcc agtgacaagt
     5941 ctgtctgcct attcaccgat tttgattctc aaacaaatgt gtcacaaagt aaggattctg
     6001 atgtgtatat cacagacaaa actgtgctag acatgaggtc tatggacttc aagagcaaca
     6061 gtgctgtggc ctggagcaac aaatctgact ttgcatgtgc aaacgccttc aacaacagca
     6121 ttattccaga agacaccttc ttccccagcc caggtaaggg cagctttggt gccttcgcag
     6181 gctgtttcct tgcttcagga atggccatcc tctagagtcg acctgcaggc atgcaagctt
     6241 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     6301 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     6361 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     6421 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     6481 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6541 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6601 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6661 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6721 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6781 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6841 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6901 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6961 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7021 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7081 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7141 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7201 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caa
//
