LOCUS       sequence-412256-        5650 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 5650)
  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 5650)
  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-602_NY-ESO-1_TCR_RFP
FEATURES             Location/Qualifiers
     source          1..5650
                     /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             2052..2756
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2194..2212)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2462..2481
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2820..2873
                     /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             3831..3887
                     /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(4660..4676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4660..4676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4673..4695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4684..4700
                     /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(4708..4714,4715..4732,4733..4738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4891..4908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5062..5650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5142..5161)
                     /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 ggcggtggtg
     1981 gtagtggagg gcagtgcacc aactatgcgc tgctgaaact ggcgggcgat gtggaaagca
     2041 acccgggccc ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     2101 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggtgagg
     2161 gagagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     2221 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     2281 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     2341 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     2401 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2461 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     2521 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2581 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     2641 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     2701 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagaggg
     2761 ctaagagagg ttcaggatct ggtggtggta gtgggggagg aaccggtgct ggaagtggtg
     2821 agggcagggg atctctcctt acttgtggcg acgtggagga gaaccccggc cccatgagca
     2881 tcggcctcct gtgctgtgca gccttgtctc tcctgtgggc aggtccagtg aatgctggtg
     2941 tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca ctgcagtgtg
     3001 cccaggatat gaaccatgaa tacatgtcct ggtatcgaca agacccaggc atggggctga
     3061 ggctgattca ttactcagtt ggtgctggta tcactgacca aggagaagtc cccaatggct
     3121 acaatgtctc cagatcaacc acagaggatt tcccgctcag gctgctgtcg gctgctccct
     3181 cccagacatc tgtgtacttc tgtgccagca gttacgtcgg gaacaccggg gagctgtttt
     3241 ttggagaagg ctctaggctg accgtactgg aggacctgaa aaacgtgttc ccacccgagg
     3301 tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc acactggtat
     3361 gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg aatgggaagg
     3421 aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc gccctcaatg
     3481 actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg cagaaccccc
     3541 gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac gagtggaccc
     3601 aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt agagcagact
     3661 gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc ctctatgaga
     3721 tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg ctgatggcta
     3781 tggtcaagag aaaggattcc agaggccggg ccaagcggtc cggatccgga gccaccaact
     3841 tcagcctgct gaagcaggcc ggcgacgtgg aggagaaccc cggccccatg gagaccctct
     3901 tgggcctgct tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc
     3961 agattcctgc agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca
     4021 ctgatagcgc tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat
     4081 ctctgttgct tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc
     4141 tggataaatc atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag
     4201 ccacctacct ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa
     4261 gaggaaccag ccttattgtt catccgtata tccagaaccc tgaccctgcg gtgtaccagc
     4321 tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa
     4381 caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact gtgctagaca
     4441 tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa tctgactttg
     4501 catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag
     4561 gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccatcctct
     4621 agagtcgacc tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4801 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4861 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5461 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5641 aggatctcaa
//
