LOCUS       sequence-412334-        6148 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 6148)
  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 6148)
  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-858_CD19-BBz_CAR_TFAP4
FEATURES             Location/Qualifiers
     source          1..6148
                     /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             3979..4008
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4744..4800
                     /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(5171..5193)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5182..5198
                     /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(5206..5212,5213..5230,5231..5236))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5251..5272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5389..5406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5560..6148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5640..5659)
                     /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 gagaaccacg 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 gccctttcga ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagatggtc 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 cttctcctgg tgacaagcct tctgctctgt gagttaccac acccagcatt cctcctgatc
     3241 ccagacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc
     3301 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa
     3361 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca
     3421 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag
     3481 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga
     3541 ggggggacta agttggaaat aacaggctcc acctctggat ccggcaagcc cggatctggc
     3601 gagggatcca ccaagggcga ggtgaaactg caggagtcag gacctggcct ggtggcgccc
     3661 tcacagagcc tgtccgtcac atgcactgtc tcaggggtct cattacccga ctatggtgta
     3721 agctggattc gccagcctcc acgaaagggt ctggagtggc tgggagtaat atggggtagt
     3781 gaaaccacat actataattc agctctcaaa tccagactga ccatcatcaa ggacaactcc
     3841 aagagccaag ttttcttaaa aatgaacagt ctgcaaactg atgacacagc catttactac
     3901 tgtgccaaac attattacta cggtggtagc tatgctatgg actactgggg tcaaggaacc
     3961 tcagtcaccg tctcctcaga gcaaaagctc atttctgaag aggacttgtt cgtgccggtc
     4021 ttcctgccag cgaagcccac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc
     4081 atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca
     4141 gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg
     4201 acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaacca caggaacaaa
     4261 cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact
     4321 actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa
     4381 ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag
     4441 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt
     4501 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac
     4561 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag
     4621 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac
     4681 acctacgacg cccttcacat gcaggccctg ccccctcgcc gggccaagcg gtccggatcc
     4741 ggagccacca acttcagcct gctgaagcag gccggcgacg tggaggagaa ccccggcccc
     4801 gcggtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat
     4861 tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa
     4921 actgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac
     4981 aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga agacaccttc
     5041 ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct tgcttcagga
     5101 atggccaggg gatcctctag agtcgacctg caggcatgca agcttggcgt aatcatggta
     5161 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5221 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6001 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6121 gattacgcgc agaaaaaaag gatctcaa
//
