LOCUS       sequence-412290-        6814 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 6814)
  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 6814)
  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-749_HA-GD2-28z_CAR_TFAP4
FEATURES             Location/Qualifiers
     source          1..6814
                     /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             3124..3177
                     /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             5410..5466
                     /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(5824..5840)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5824..5840)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5837..5859)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5848..5864
                     /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(5872..5878,5879..5896,5897..5902))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5917..5938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6055..6072)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6226..6814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6306..6325)
                     /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 tggaggatct
     1981 ggaggacagt gcaccaacta tgcgctgctg aaactggcgg gcgatgtgga aagcaacccg
     2041 ggcccgatgg agtatttcat ggtgcccact cagaaggtgc cctctttgca acatttcagg
     2101 aaaacagaga aagaagtgat aggagggctc tgtagccttg ccaacattcc actaaccccc
     2161 gagactcagc gggaccagga gcggcggatt cggcgggaga tcgccaacag caacgagcgg
     2221 agacgcatgc agagcatcaa cgcgggattc cagtccctca agaccctcat cccccacaca
     2281 gacggagaga agctcagcaa ggcagccatt ctccagcaga cagccgagta catcttctcc
     2341 ctggagcagg agaagaccag gctcttgcag cagaacacac agctcaagcg cttcatccag
     2401 gagctgagcg gctcgtcccc caagcgacgg cgggcagagg acaaggacga aggcataggc
     2461 tccccggaca tctgggagga cgagaaggcg gaggacctgc ggcgggagat gattgagctg
     2521 cggcagcagc tggacaagga gcgctcggtg cgcatgatgc tggaggagca ggtgcgctcg
     2581 ctggaggccc acatgtaccc ggaaaagctc aaggtgattg cgcagcaggt gcagctgcag
     2641 cagcagcagg aacaggtgag gctgctgcac caggagaagc tggagcggga acagcagcag
     2701 ctgcggaccc agcttctgcc ccctccggcc cccacccacc accccacggt gatcgtgcca
     2761 gcaccgcctc ctcctccctc ccaccacatc aatgtcgtca ccatgggccc ctcctcggtc
     2821 atcaactctg tttccacatc ccggcaaaat ctggacacca tcgtgcaggc aatccagcac
     2881 atcgagggca cccaggaaaa gcaggagctg gaggaggagc agcggcgagc tgtcatcgtg
     2941 aagcctgtcc gcagctgccc ggaggccccc acctctgaca ccgcctccga ctccgaggcc
     3001 tcagacagtg acgccatgga ccagagccgg gaggagccgt cgggggacgg ggagcttccc
     3061 agggctaaga gaggttcagg atctggaggc ggatctgggg gaggaaccgg tgctggaagt
     3121 ggtgagggca ggggatctct ccttacttgt ggcgacgtgg aggagaaccc cggccccatg
     3181 gaattcggcc tgagctggct gttcctggtg gccatcctga agggcgtgca gtgcagcaga
     3241 gacatcctgc tgacccagac ccctctgagc ctgcctgtgt ctctgggcga tcaggccagc
     3301 atcagctgca gatccagcca gagcctggtg caccggaacg gcaacaccta cctgcactgg
     3361 tatctgcaga agcccggcca gagccccaag ctgctgatcc acaaggtgtc caaccggttc
     3421 agcggcgtgc ccgacagatt ttctggcagc ggctccggca ccgacttcac cctgaagatc
     3481 agccgggtgg aagccgagga cctgggcgtg tacttctgca gccagtccac ccacgtgccc
     3541 cccctgacat ttggcgccgg aacaaagctg gaactgaagg ggggaggcgg atctggcggc
     3601 ggaggaagtg gcggaggggg atctgaagtg aagctgcagc agtccggccc cagcctggtg
     3661 gaacctggcg cctctgtgat gatctcctgc aaggccagcg gcagctcctt caccggctac
     3721 aacatgaact gggtgcgcca gaacatcggc aagagcctgg aatggatcgg cgccatcgac
     3781 ccctactacg gcggcaccag ctacaaccag aagttcaagg gcagagccac cctgaccgtg
     3841 gacaagagca gcagcaccgc ctacatgcac ctgaagtccc tgaccagcga ggacagcgcc
     3901 gtgtactact gcgtgtccgg catgaagtac tggggccagg gcacaagcgt gaccgtgtct
     3961 agcgccaaga ccaccccccc tagcgtgtac ggaagagtga cagtgtcctc tgccgagccc
     4021 aagagctgcg acaagaccca cacctgtccc ccttgtcctg cccctcctgt tgcaggccct
     4081 tccgtgttcc tgttcccccc aaagcccaag gacacactga tgatcagcag aacccccgaa
     4141 gtgacctgcg tggtggtgga cgtgtcccac gaggacccag aagtgaagtt caattggtac
     4201 gtggacggcg tggaagtgca caacgccaag acaaagccca gagaggaaca gtaccaaagc
     4261 acctaccggg tggtgtccgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagag
     4321 tacaagtgca aagtgtccaa caaggccctg cctgccccca tcgagaaaac catcagcaag
     4381 gccaagggcc agccccgcga accccaggtg tacacactgc cccctagcag ggacgagctg
     4441 accaagaacc aggtgtccct gacatgcctc gtgaagggct tctacccctc cgatatcgcc
     4501 gtggaatggg agagcaacgg ccagcccgag aacaactaca agacaacccc tcccgtgctg
     4561 gacagcgacg gctcattctt cctgtacagc aagctgacag tggataagtc ccggtggcag
     4621 cagggcaacg tgttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag
     4681 aaaagcctgt ccctgagccc cggcaagaag gaccccaaag cggccgcaat tgaagttatg
     4741 tatcctcctc cttacctaga caatgagaag agcaatggaa ccattatcca tgtgaaaggg
     4801 aaacaccttt gtccaagtcc cctatttccc ggaccttcta agcccttttg ggtgctggtg
     4861 gtggttgggg gagtcctggc ttgctatagc ttgctagtaa cagtggcctt tattattttc
     4921 tgggtgagga gtaagaggag caggctcctg cacagtgact acatgaacat gactccccgc
     4981 cgccccgggc ccacccgcaa gcattaccag ccctatgccc caccacgcga cttcgcagcc
     5041 tatcgctcca gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag
     5101 aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag
     5161 agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc
     5221 ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa
     5281 ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc
     5341 aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgccgggc caagcggtcc
     5401 ggatccggag ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc
     5461 ggccccgcgg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc
     5521 accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca
     5581 gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg
     5641 agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac
     5701 accttcttcc ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct
     5761 tcaggaatgg ccaggggatc ctctagagtc gacctgcagg catgcaagct tggcgtaatc
     5821 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5881 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5941 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     6001 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6061 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6121 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     6181 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6241 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6301 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6361 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6421 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6481 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6541 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6601 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6661 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6721 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     6781 gcagcagatt acgcgcagaa aaaaaggatc tcaa
//
