LOCUS       sequence-412292-        7272 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 7272)
  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 7272)
  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-804_HA-GD2-28z_CAR_BATF-TFAP4
FEATURES             Location/Qualifiers
     source          1..7272
                     /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             1948..2007
                     /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             2485..2544
                     /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             3583..3636
                     /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             5869..5925
                     /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(6282..6298)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6282..6298)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6295..6317)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6306..6322
                     /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(6330..6336,6337..6354,6355..6360))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6375..6396
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6513..6530)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6684..7272)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6764..6783)
                     /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 ccctgcccag tgcaccaact atgcgctgct gaaactggcg
     1981 ggcgatgtgg aaagcaaccc gggcccgatg cctcacagct ccgacagcag tgactccagc
     2041 ttcagccgct ctcctccccc tggcaaacag gactcatctg atgatgtgag aagagttcag
     2101 aggagggaga aaaatcgtat tgccgcccag aagagccgac agaggcagac acagaaggcc
     2161 gacaccctgc acctggagag cgaagacctg gagaaacaga acgcggctct acgcaaggag
     2221 atcaagcagc tcacagagga actgaagtac ttcacgtcgg tgctgaacag ccacgagccc
     2281 ctgtgctcgg tgctggccgc cagcacgccc tcgccccccg aggtggtgta cagcgcccac
     2341 gcattccacc aacctcatgt cagctccccg cgcttccagc ccagggctaa gagaggttca
     2401 ggatctggcg ggggctcagg gggaggaacc ggtgctggaa gtggtggatc gggtgggact
     2461 agtggcggtg gaggatctgg aggacagtgc accaactatg cgctgctgaa actggcgggc
     2521 gatgtggaaa gcaacccggg cccgatggag tatttcatgg tgcccactca gaaggtgccc
     2581 tctttgcaac atttcaggaa aacagagaaa gaagtgatag gagggctctg tagccttgcc
     2641 aacattccac taacccccga gactcagcgg gaccaggagc ggcggattcg gcgggagatc
     2701 gccaacagca acgagcggag acgcatgcag agcatcaacg cgggattcca gtccctcaag
     2761 accctcatcc cccacacaga cggagagaag ctcagcaagg cagccattct ccagcagaca
     2821 gccgagtaca tcttctccct ggagcaggag aagaccaggc tcttgcagca gaacacacag
     2881 ctcaagcgct tcatccagga gctgagcggc tcgtccccca agcgacggcg ggcagaggac
     2941 aaggacgaag gcataggctc cccggacatc tgggaggacg agaaggcgga ggacctgcgg
     3001 cgggagatga ttgagctgcg gcagcagctg gacaaggagc gctcggtgcg catgatgctg
     3061 gaggagcagg tgcgctcgct ggaggcccac atgtacccgg aaaagctcaa ggtgattgcg
     3121 cagcaggtgc agctgcagca gcagcaggaa caggtgaggc tgctgcacca ggagaagctg
     3181 gagcgggaac agcagcagct gcggacccag cttctgcccc ctccggcccc cacccaccac
     3241 cccacggtga tcgtgccagc accgcctcct cctccctccc accacatcaa tgtcgtcacc
     3301 atgggcccct cctcggtcat caactctgtt tccacatccc ggcaaaatct ggacaccatc
     3361 gtgcaggcaa tccagcacat cgagggcacc caggaaaagc aggagctgga ggaggagcag
     3421 cggcgagctg tcatcgtgaa gcctgtccgc agctgcccgg aggcccccac ctctgacacc
     3481 gcctccgact ccgaggcctc agacagtgac gccatggacc agagccggga ggagccgtcg
     3541 ggggacgggg agcttcccag ggctaagaga ggttcaggat ctgagggcag gggatctctc
     3601 cttacttgtg gcgacgtgga ggagaacccc ggccccatgg aattcggcct gagctggctg
     3661 ttcctggtgg ccatcctgaa gggcgtgcag tgcagcagag acatcctgct gacccagacc
     3721 cctctgagcc tgcctgtgtc tctgggcgat caggccagca tcagctgcag atccagccag
     3781 agcctggtgc accggaacgg caacacctac ctgcactggt atctgcagaa gcccggccag
     3841 agccccaagc tgctgatcca caaggtgtcc aaccggttca gcggcgtgcc cgacagattt
     3901 tctggcagcg gctccggcac cgacttcacc ctgaagatca gccgggtgga agccgaggac
     3961 ctgggcgtgt acttctgcag ccagtccacc cacgtgcccc ccctgacatt tggcgccgga
     4021 acaaagctgg aactgaaggg gggaggcgga tctggcggcg gaggaagtgg cggaggggga
     4081 tctgaagtga agctgcagca gtccggcccc agcctggtgg aacctggcgc ctctgtgatg
     4141 atctcctgca aggccagcgg cagctccttc accggctaca acatgaactg ggtgcgccag
     4201 aacatcggca agagcctgga atggatcggc gccatcgacc cctactacgg cggcaccagc
     4261 tacaaccaga agttcaaggg cagagccacc ctgaccgtgg acaagagcag cagcaccgcc
     4321 tacatgcacc tgaagtccct gaccagcgag gacagcgccg tgtactactg cgtgtccggc
     4381 atgaagtact ggggccaggg cacaagcgtg accgtgtcta gcgccaagac caccccccct
     4441 agcgtgtacg gaagagtgac agtgtcctct gccgagccca agagctgcga caagacccac
     4501 acctgtcccc cttgtcctgc ccctcctgtt gcaggccctt ccgtgttcct gttcccccca
     4561 aagcccaagg acacactgat gatcagcaga acccccgaag tgacctgcgt ggtggtggac
     4621 gtgtcccacg aggacccaga agtgaagttc aattggtacg tggacggcgt ggaagtgcac
     4681 aacgccaaga caaagcccag agaggaacag taccaaagca cctaccgggt ggtgtccgtg
     4741 ctgaccgtgc tgcatcagga ttggctgaac ggcaaagagt acaagtgcaa agtgtccaac
     4801 aaggccctgc ctgcccccat cgagaaaacc atcagcaagg ccaagggcca gccccgcgaa
     4861 ccccaggtgt acacactgcc ccctagcagg gacgagctga ccaagaacca ggtgtccctg
     4921 acatgcctcg tgaagggctt ctacccctcc gatatcgccg tggaatggga gagcaacggc
     4981 cagcccgaga acaactacaa gacaacccct cccgtgctgg acagcgacgg ctcattcttc
     5041 ctgtacagca agctgacagt ggataagtcc cggtggcagc agggcaacgt gttcagctgc
     5101 tccgtgatgc acgaggccct gcacaaccac tacacccaga aaagcctgtc cctgagcccc
     5161 ggcaagaagg accccaaagc ggccgcaatt gaagttatgt atcctcctcc ttacctagac
     5221 aatgagaaga gcaatggaac cattatccat gtgaaaggga aacacctttg tccaagtccc
     5281 ctatttcccg gaccttctaa gcccttttgg gtgctggtgg tggttggggg agtcctggct
     5341 tgctatagct tgctagtaac agtggccttt attattttct gggtgaggag taagaggagc
     5401 aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag
     5461 cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc
     5521 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc
     5581 aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag
     5641 atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa
     5701 gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag
     5761 gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt
     5821 cacatgcagg ccctgccccc tcgccgggcc aagcggtccg gatccggagc caccaacttc
     5881 agcctgctga agcaggccgg cgacgtggag gagaaccccg gccccgcggt gtaccagctg
     5941 agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca
     6001 aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg
     6061 aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca
     6121 tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccaggt
     6181 aagggcagct ttggtgcctt cgcaggctgt ttccttgctt caggaatggc caggggatcc
     6241 tctagatcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     6301 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     6361 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     6421 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6481 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6541 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6601 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6661 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6721 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6781 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6841 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6901 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6961 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7021 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7081 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     7141 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     7201 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     7261 aaaggatctc aa
//
