LOCUS       sequence-412300-        7581 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 7581)
  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 7581)
  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-755_HA-GD2-28z_CAR_RFP-JUN
FEATURES             Location/Qualifiers
     source          1..7581
                     /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             3892..3945
                     /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             6178..6234
                     /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(6591..6607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6591..6607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6604..6626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6615..6631
                     /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(6639..6645,6646..6663,6664..6669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6684..6705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6822..6839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6993..7581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7073..7092)
                     /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 agtggcggtg gaggctcggg aggacagtgc
     2821 accaactatg cgctgctgaa actggcgggc gatgtggaaa gcaacccggg cccgatgacc
     2881 gcgaaaatgg agacaacatt ttacgatgac gctcttaatg cgtcctttct gccatcagaa
     2941 tccggcccat acggatatag caatccgaaa atattgaagc aatctatgac attgaatctt
     3001 gctgatcctg tcggttcctt gaaacctcat ctgcgggcaa agaattccga tctgttgact
     3061 agcccagatg taggactcct gaaactcgca tcccctgaac tcgaacggct cattatacaa
     3121 tcaagtaatg gacatattac gacgacccct actcctacgc aatttctttg tcctaagaat
     3181 gtcaccgacg aacaagaagg ttttgcagaa gggtttgtac gggcattggc ggagcttcat
     3241 tcacaaaata ccctgcctag tgtgacctct gccgctcaac cagtgaatgg agctggaatg
     3301 gtcgcccctg ccgttgcaag cgttgccggc ggaagtggat ctggcgggtt tagtgcatca
     3361 cttcattctg aaccaccagt gtatgccaat ctgtccaatt ttaatcccgg agccctctca
     3421 tctggaggag gcgccccttc ttatggtgca gcgggacttg cgttccctgc acagccacaa
     3481 caacaacaac aaccccctca tcatttgcca caacaaatgc ctgtccaaca tccacgcctg
     3541 caagctctca aagaagaacc ccaaaccgtc cctgaaatgc caggggaaac cccaccactt
     3601 tcaccgattg atatggaatc tcaagaaaga ataaaagccg aaagaaaacg tatgcgcaat
     3661 cggatagccg cgtcaaaatg tcgcaagcgc aaactcgaac ggattgcacg cttggaagag
     3721 aaggttaaga ctctgaaggc gcaaaattct gaacttgctt caaccgctaa tatgctgcga
     3781 gagcaagtcg cccaattgaa gcaaaaggta atgaatcatg tgaatagcgg atgtcagctg
     3841 atgctcactc aacaactgca gactttcagg gctaagagag gttcaggatc tgagggcagg
     3901 ggatctctcc ttacttgtgg cgacgtggag gagaaccccg gccccatgga attcggcctg
     3961 agctggctgt tcctggtggc catcctgaag ggcgtgcagt gcagcagaga catcctgctg
     4021 acccagaccc ctctgagcct gcctgtgtct ctgggcgatc aggccagcat cagctgcaga
     4081 tccagccaga gcctggtgca ccggaacggc aacacctacc tgcactggta tctgcagaag
     4141 cccggccaga gccccaagct gctgatccac aaggtgtcca accggttcag cggcgtgccc
     4201 gacagatttt ctggcagcgg ctccggcacc gacttcaccc tgaagatcag ccgggtggaa
     4261 gccgaggacc tgggcgtgta cttctgcagc cagtccaccc acgtgccccc cctgacattt
     4321 ggcgccggaa caaagctgga actgaagggg ggaggcggat ctggcggcgg aggaagtggc
     4381 ggagggggat ctgaagtgaa gctgcagcag tccggcccca gcctggtgga acctggcgcc
     4441 tctgtgatga tctcctgcaa ggccagcggc agctccttca ccggctacaa catgaactgg
     4501 gtgcgccaga acatcggcaa gagcctggaa tggatcggcg ccatcgaccc ctactacggc
     4561 ggcaccagct acaaccagaa gttcaagggc agagccaccc tgaccgtgga caagagcagc
     4621 agcaccgcct acatgcacct gaagtccctg accagcgagg acagcgccgt gtactactgc
     4681 gtgtccggca tgaagtactg gggccagggc acaagcgtga ccgtgtctag cgccaagacc
     4741 acccccccta gcgtgtacgg aagagtgaca gtgtcctctg ccgagcccaa gagctgcgac
     4801 aagacccaca cctgtccccc ttgtcctgcc cctcctgttg caggcccttc cgtgttcctg
     4861 ttccccccaa agcccaagga cacactgatg atcagcagaa cccccgaagt gacctgcgtg
     4921 gtggtggacg tgtcccacga ggacccagaa gtgaagttca attggtacgt ggacggcgtg
     4981 gaagtgcaca acgccaagac aaagcccaga gaggaacagt accaaagcac ctaccgggtg
     5041 gtgtccgtgc tgaccgtgct gcatcaggat tggctgaacg gcaaagagta caagtgcaaa
     5101 gtgtccaaca aggccctgcc tgcccccatc gagaaaacca tcagcaaggc caagggccag
     5161 ccccgcgaac cccaggtgta cacactgccc cctagcaggg acgagctgac caagaaccag
     5221 gtgtccctga catgcctcgt gaagggcttc tacccctccg atatcgccgt ggaatgggag
     5281 agcaacggcc agcccgagaa caactacaag acaacccctc ccgtgctgga cagcgacggc
     5341 tcattcttcc tgtacagcaa gctgacagtg gataagtccc ggtggcagca gggcaacgtg
     5401 ttcagctgct ccgtgatgca cgaggccctg cacaaccact acacccagaa aagcctgtcc
     5461 ctgagccccg gcaagaagga ccccaaagcg gccgcaattg aagttatgta tcctcctcct
     5521 tacctagaca atgagaagag caatggaacc attatccatg tgaaagggaa acacctttgt
     5581 ccaagtcccc tatttcccgg accttctaag cccttttggg tgctggtggt ggttggggga
     5641 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt
     5701 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc
     5761 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga
     5821 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat
     5881 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg
     5941 gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa
     6001 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg
     6061 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac
     6121 gacgcccttc acatgcaggc cctgccccct cgccgggcca agcggtccgg atccggagcc
     6181 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccgcggtg
     6241 taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat
     6301 tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg
     6361 ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct
     6421 gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc
     6481 agcccaggta agggcagctt tggtgccttc gcaggctgtt tccttgcttc aggaatggcc
     6541 aggggatcct ctagatcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     6601 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     6661 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     6721 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6781 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     6841 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     6901 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     6961 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     7021 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     7081 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     7141 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     7201 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     7261 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     7321 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     7381 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     7441 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     7501 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     7561 cgcagaaaaa aaggatctca a
//
