LOCUS       sequence-412338-        6615 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 6615)
  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 6615)
  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-874_HA-GD2-28z_CAR_IL2RA
FEATURES             Location/Qualifiers
     source          1..6615
                     /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             1987..2046
                     /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             2926..2979
                     /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             5212..5268
                     /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(5625..5641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5625..5641)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5638..5660)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5649..5665
                     /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(5673..5679,5680..5697,5698..5703))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5718..5739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5856..5873)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6027..6615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6107..6126)
                     /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 cattcgccat tcaggctgcg caactgttgg
     1501 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1561 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1621 gccagtgaat tcgagctcgg taccctttca ggtttccttg agtggcaggc caggcctggc
     1681 cgtgaacgtt cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct
     1741 gagtcccagt ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga
     1801 gcacgtgcat tcgcacgccc acagagcccc gcccttgtcc atcactggca tctggactcc
     1861 agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc
     1921 acagatatcc agaaccctga ccctgccgga tcgggtggga ctagtggcgg gggtgggagc
     1981 ggtggacagt gcaccaacta tgcgctgctg aaactggcgg gcgatgtgga aagcaacccg
     2041 ggcccgatgg attcatacct gctgatgtgg ggactgctca cgttcatcat ggtgcctggc
     2101 tgccaggcag agctctgtga cgatgacccg ccagagatcc cacacgccac attcaaagcc
     2161 atggcctaca aggaaggaac catgttgaac tgtgaatgca agagaggttt ccgcagaata
     2221 aaaagcgggt cactctatat gctctgtaca ggaaactcta gccactcgtc ctgggacaac
     2281 caatgtcaat gcacaagctc tgccactcgg aacacaacga aacaagtgac acctcaacct
     2341 gaagaacaga aagaaaggaa aaccacagaa atgcaaagtc caatgcagcc agtggaccaa
     2401 gcgagccttc caggtcactg cagggaacct ccaccatggg aaaatgaagc cacagagaga
     2461 atttatcatt tcgtggtggg gcagatggtt tattatcagt gcgtccaggg atacagggct
     2521 ctacacagag gtcctgctga gagcgtctgc aaaatgaccc acgggaagac aaggtggacc
     2581 cagccccagc tcatatgcac aggtgaaatg gagaccagtc agtttccagg tgaagagaag
     2641 cctcaggcaa gccccgaagg ccgtcctgag agtgagactt cctgcctcgt cacaacaaca
     2701 gattttcaaa tacagacaga aatggctgca accatggaga cgtccatatt tacaacagag
     2761 taccaggtag cagtggccgg ctgtgttttc ctgctgatca gcgtcctcct cctgagtggg
     2821 ctcacctggc agcggagaca gaggaagagt agaagaacaa tcagggctaa gagaggttca
     2881 ggatctggtg gaggttcggg agggggaacc ggtgctggaa gtggtgaggg caggggatct
     2941 ctccttactt gtggcgacgt ggaggagaac cccggcccca tggaattcgg cctgagctgg
     3001 ctgttcctgg tggccatcct gaagggcgtg cagtgcagca gagacatcct gctgacccag
     3061 acccctctga gcctgcctgt gtctctgggc gatcaggcca gcatcagctg cagatccagc
     3121 cagagcctgg tgcaccggaa cggcaacacc tacctgcact ggtatctgca gaagcccggc
     3181 cagagcccca agctgctgat ccacaaggtg tccaaccggt tcagcggcgt gcccgacaga
     3241 ttttctggca gcggctccgg caccgacttc accctgaaga tcagccgggt ggaagccgag
     3301 gacctgggcg tgtacttctg cagccagtcc acccacgtgc cccccctgac atttggcgcc
     3361 ggaacaaagc tggaactgaa ggggggaggc ggatctggcg gcggaggaag tggcggaggg
     3421 ggatctgaag tgaagctgca gcagtccggc cccagcctgg tggaacctgg cgcctctgtg
     3481 atgatctcct gcaaggccag cggcagctcc ttcaccggct acaacatgaa ctgggtgcgc
     3541 cagaacatcg gcaagagcct ggaatggatc ggcgccatcg acccctacta cggcggcacc
     3601 agctacaacc agaagttcaa gggcagagcc accctgaccg tggacaagag cagcagcacc
     3661 gcctacatgc acctgaagtc cctgaccagc gaggacagcg ccgtgtacta ctgcgtgtcc
     3721 ggcatgaagt actggggcca gggcacaagc gtgaccgtgt ctagcgccaa gaccaccccc
     3781 cctagcgtgt acggaagagt gacagtgtcc tctgccgagc ccaagagctg cgacaagacc
     3841 cacacctgtc ccccttgtcc tgcccctcct gttgcaggcc cttccgtgtt cctgttcccc
     3901 ccaaagccca aggacacact gatgatcagc agaacccccg aagtgacctg cgtggtggtg
     3961 gacgtgtccc acgaggaccc agaagtgaag ttcaattggt acgtggacgg cgtggaagtg
     4021 cacaacgcca agacaaagcc cagagaggaa cagtaccaaa gcacctaccg ggtggtgtcc
     4081 gtgctgaccg tgctgcatca ggattggctg aacggcaaag agtacaagtg caaagtgtcc
     4141 aacaaggccc tgcctgcccc catcgagaaa accatcagca aggccaaggg ccagccccgc
     4201 gaaccccagg tgtacacact gccccctagc agggacgagc tgaccaagaa ccaggtgtcc
     4261 ctgacatgcc tcgtgaaggg cttctacccc tccgatatcg ccgtggaatg ggagagcaac
     4321 ggccagcccg agaacaacta caagacaacc cctcccgtgc tggacagcga cggctcattc
     4381 ttcctgtaca gcaagctgac agtggataag tcccggtggc agcagggcaa cgtgttcagc
     4441 tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaaaagcct gtccctgagc
     4501 cccggcaaga aggaccccaa agcggccgca attgaagtta tgtatcctcc tccttaccta
     4561 gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt
     4621 cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg
     4681 gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg
     4741 agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc
     4801 aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag
     4861 ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag
     4921 ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct
     4981 gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag
     5041 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc
     5101 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc
     5161 cttcacatgc aggccctgcc ccctcgccgg gccaagcggt ccggatccgg agccaccaac
     5221 ttcagcctgc tgaagcaggc cggcgacgtg gaggagaacc ccggccccgc ggtgtaccag
     5281 ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa
     5341 acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac
     5401 atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt
     5461 gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca
     5521 ggtaagggca gctttggtgc cttcgcaggc tgtttccttg cttcaggaat ggccagggga
     5581 tcctctagat cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     5641 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     5701 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     5761 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     5821 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5881 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5941 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6001 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6061 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6121 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6181 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6241 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6301 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6361 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6421 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6481 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6541 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6601 aaaaaaggat ctcaa
//
