LOCUS       sequence-412302-        5662 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 5662)
  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 5662)
  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-832_NY-ESO-1_TCR_FAS/4-1BB
FEATURES             Location/Qualifiers
     source          1..5662
                     /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             2832..2885
                     /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             3843..3899
                     /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(4672..4688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4672..4688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4685..4707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4696..4712
                     /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(4720..4726,4727..4744,4745..4750))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4765..4786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4903..4920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5074..5662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5154..5173)
                     /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 ggcgggggcg
     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 aaaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg agaccagtac
     2701 aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa gaaggaggat
     2761 gtgaactgag ggctaagaga ggttcaggat ctggcggcgg gtctggcgga ggaaccggtg
     2821 ctggaagtgg tgagggcagg ggatctctcc ttacttgtgg cgacgtggag gagaaccccg
     2881 gccccatgag catcggcctc ctgtgctgtg cagccttgtc tctcctgtgg gcaggtccag
     2941 tgaatgctgg tgtcactcag accccaaaat tccaggtcct gaagacagga cagagcatga
     3001 cactgcagtg tgcccaggat atgaaccatg aatacatgtc ctggtatcga caagacccag
     3061 gcatggggct gaggctgatt cattactcag ttggtgctgg tatcactgac caaggagaag
     3121 tccccaatgg ctacaatgtc tccagatcaa ccacagagga tttcccgctc aggctgctgt
     3181 cggctgctcc ctcccagaca tctgtgtact tctgtgccag cagttacgtc gggaacaccg
     3241 gggagctgtt ttttggagaa ggctctaggc tgaccgtact ggaggacctg aaaaacgtgt
     3301 tcccacccga ggtcgctgtg tttgagccat cagaagcaga gatctcccac acccaaaagg
     3361 ccacactggt atgcctggcc acaggcttct accccgacca cgtggagctg agctggtggg
     3421 tgaatgggaa ggaggtgcac agtggggtca gcacagaccc gcagcccctc aaggagcagc
     3481 ccgccctcaa tgactccaga tactgcctga gcagccgcct gagggtctcg gccaccttct
     3541 ggcagaaccc ccgcaaccac ttccgctgtc aagtccagtt ctacgggctc tcggagaatg
     3601 acgagtggac ccaggatagg gccaaacccg tcacccagat cgtcagcgcc gaggcctggg
     3661 gtagagcaga ctgtggcttc acctccgagt cttaccagca aggggtcctg tctgccacca
     3721 tcctctatga gatcttgcta gggaaggcca ccttgtatgc cgtgctggtc agtgccctcg
     3781 tgctgatggc tatggtcaag agaaaggatt ccagaggccg ggccaagcgg tccggatccg
     3841 gagccaccaa cttcagcctg ctgaagcagg ccggcgacgt ggaggagaac cccggcccca
     3901 tggagaccct cttgggcctg cttatccttt ggctgcagct gcaatgggtg agcagcaaac
     3961 aggaggtgac gcagattcct gcagctctga gtgtcccaga aggagaaaac ttggttctca
     4021 actgcagttt cactgatagc gctatttaca acctccagtg gtttaggcag gaccctggga
     4081 aaggtctcac atctctgttg cttattcagt caagtcagag agagcaaaca agtggaagac
     4141 ttaatgcctc gctggataaa tcatcaggac gtagtacttt atacattgca gcttctcagc
     4201 ctggtgactc agccacctac ctctgtgctg tgaggcccct gtacggagga agctacatac
     4261 ctacatttgg aagaggaacc agccttattg ttcatccgta tatccagaac cctgaccctg
     4321 cggtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt
     4381 ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa
     4441 ctgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc tggagcaaca
     4501 aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa gacaccttct
     4561 tccccagccc aggtaagggc agctttggtg ccttcgcagg ctgtttcctt gcttcaggaa
     4621 tggccatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     4681 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4741 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4801 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4861 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4921 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4981 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5041 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5101 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5161 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5221 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5281 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5341 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5401 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5461 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5521 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5581 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5641 gcgcagaaaa aaaggatctc aa
//
