LOCUS       sequence-412282-        7419 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 7419)
  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 7419)
  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-727_HA-GD2-28z_CAR_RFP-tNGFR
FEATURES             Location/Qualifiers
     source          1..7419
                     /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             2008..2712
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2150..2168)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2418..2437
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2815..2874
                     /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             3730..3783
                     /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             6016..6072
                     /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(6429..6445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6429..6445)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6442..6464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6453..6469
                     /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(6477..6483,6484..6501,6502..6507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6522..6543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6660..6677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6831..7419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6911..6930)
                     /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 gggcccggtg agcaagggcg aggaggataa catggccatc
     2041 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     2101 gagatcgagg gtgagggaga gggccgcccc tacgagggca cccagaccgc caagctgaag
     2161 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     2221 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     2281 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     2341 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     2401 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     2461 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     2521 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     2581 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     2641 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     2701 gagctgtaca agagggctaa gagaggttca ggatctggtg gtggtagtgg gggaggaacc
     2761 ggtgctggaa gtggtggatc gggtgggact agtggcggcg gcggttctgg tggtcagtgc
     2821 accaactatg cgctgctgaa actggcgggc gatgtggaaa gcaacccggg cccgatgggc
     2881 gctggtgcaa ctggacgggc catggatgga ccacggcttt tgctcctgtt gttgttgggc
     2941 gtcagccttg gaggggctaa agaggcctgc ccgaccggtt tgtacacaca ctccggcgag
     3001 tgttgtaagg cgtgtaatct tggcgaaggg gtcgcccaac cgtgtggcgc caaccagacg
     3061 gtttgtgagc cttgcttgga tagcgtcacg ttttccgacg tcgtttcagc caccgaacca
     3121 tgcaaacctt gtacggaatg cgtgggtctg cagagcatga gcgcaccgtg cgtagaggcg
     3181 gacgatgcgg tatgccgatg tgcctacggc tactaccaag acgagacgac agggaggtgt
     3241 gaggcatgca gagtatgtga ggccgggtct ggactcgtat tttcctgtca agacaaacaa
     3301 aacacagtat gtgaggagtg cccggatggg acttattccg atgaagcgaa tcatgtggac
     3361 ccgtgcttgc catgtacggt ctgtgaggat accgaaagac agctccgaga atgcacccgc
     3421 tgggctgacg cggaatgcga agaaatccca ggtcgatgga tcacccgctc aacccccccc
     3481 gaagggtcag attctacggc gccctctact caggagccag aagctccacc cgaacaagac
     3541 ctgatcgctt ctacggtggc aggtgtcgtt acaacagtca tgggttcctc acaacctgtg
     3601 gttacgcggg ggacaactga caatttgatt ccagtatatt gtagcattct ggccgcggtc
     3661 gtggtagggc ttgtcgcgta tatagcgttc aaaaggtgga acagcagggc taagagaggt
     3721 tcaggatctg agggcagggg atctctcctt acttgtggcg acgtggagga gaaccccggc
     3781 cccatggaat tcggcctgag ctggctgttc ctggtggcca tcctgaaggg cgtgcagtgc
     3841 agcagagaca tcctgctgac ccagacccct ctgagcctgc ctgtgtctct gggcgatcag
     3901 gccagcatca gctgcagatc cagccagagc ctggtgcacc ggaacggcaa cacctacctg
     3961 cactggtatc tgcagaagcc cggccagagc cccaagctgc tgatccacaa ggtgtccaac
     4021 cggttcagcg gcgtgcccga cagattttct ggcagcggct ccggcaccga cttcaccctg
     4081 aagatcagcc gggtggaagc cgaggacctg ggcgtgtact tctgcagcca gtccacccac
     4141 gtgccccccc tgacatttgg cgccggaaca aagctggaac tgaagggggg aggcggatct
     4201 ggcggcggag gaagtggcgg agggggatct gaagtgaagc tgcagcagtc cggccccagc
     4261 ctggtggaac ctggcgcctc tgtgatgatc tcctgcaagg ccagcggcag ctccttcacc
     4321 ggctacaaca tgaactgggt gcgccagaac atcggcaaga gcctggaatg gatcggcgcc
     4381 atcgacccct actacggcgg caccagctac aaccagaagt tcaagggcag agccaccctg
     4441 accgtggaca agagcagcag caccgcctac atgcacctga agtccctgac cagcgaggac
     4501 agcgccgtgt actactgcgt gtccggcatg aagtactggg gccagggcac aagcgtgacc
     4561 gtgtctagcg ccaagaccac cccccctagc gtgtacggaa gagtgacagt gtcctctgcc
     4621 gagcccaaga gctgcgacaa gacccacacc tgtccccctt gtcctgcccc tcctgttgca
     4681 ggcccttccg tgttcctgtt ccccccaaag cccaaggaca cactgatgat cagcagaacc
     4741 cccgaagtga cctgcgtggt ggtggacgtg tcccacgagg acccagaagt gaagttcaat
     4801 tggtacgtgg acggcgtgga agtgcacaac gccaagacaa agcccagaga ggaacagtac
     4861 caaagcacct accgggtggt gtccgtgctg accgtgctgc atcaggattg gctgaacggc
     4921 aaagagtaca agtgcaaagt gtccaacaag gccctgcctg cccccatcga gaaaaccatc
     4981 agcaaggcca agggccagcc ccgcgaaccc caggtgtaca cactgccccc tagcagggac
     5041 gagctgacca agaaccaggt gtccctgaca tgcctcgtga agggcttcta cccctccgat
     5101 atcgccgtgg aatgggagag caacggccag cccgagaaca actacaagac aacccctccc
     5161 gtgctggaca gcgacggctc attcttcctg tacagcaagc tgacagtgga taagtcccgg
     5221 tggcagcagg gcaacgtgtt cagctgctcc gtgatgcacg aggccctgca caaccactac
     5281 acccagaaaa gcctgtccct gagccccggc aagaaggacc ccaaagcggc cgcaattgaa
     5341 gttatgtatc ctcctcctta cctagacaat gagaagagca atggaaccat tatccatgtg
     5401 aaagggaaac acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg
     5461 ctggtggtgg ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt
     5521 attttctggg tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact
     5581 ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc
     5641 gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag
     5701 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg
     5761 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag
     5821 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg
     5881 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca
     5941 gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ccgggccaag
     6001 cggtccggat ccggagccac caacttcagc ctgctgaagc aggccggcga cgtggaggag
     6061 aaccccggcc ccgcggtgta ccagctgaga gactctaaat ccagtgacaa gtctgtctgc
     6121 ctattcaccg attttgattc tcaaacaaat gtgtcacaaa gtaaggattc tgatgtgtat
     6181 atcacagaca aaactgtgct agacatgagg tctatggact tcaagagcaa cagtgctgtg
     6241 gcctggagca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag cattattcca
     6301 gaagacacct tcttccccag cccaggtaag ggcagctttg gtgccttcgc aggctgtttc
     6361 cttgcttcag gaatggccag gggatcctct agatcgacct gcaggcatgc aagcttggcg
     6421 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6481 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6541 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6601 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6661 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6721 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6781 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6841 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6901 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6961 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     7021 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     7081 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     7141 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     7201 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7261 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7321 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7381 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa
//
