LOCUS       sequence-412330-        6895 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 6895)
  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 6895)
  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-852_HA-GD2-28z_TFAP4-HA
FEATURES             Location/Qualifiers
     source          1..6895
                     /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             3061..3141
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             3205..3258
                     /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             5491..5547
                     /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(5905..5921)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5905..5921)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5918..5940)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5929..5945
                     /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(5953..5959,5960..5977,5978..5983))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5998..6019
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6136..6153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6307..6895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6387..6406)
                     /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 tacccatacg atgttccaga ttacgcttat ccttatgacg tacctgacta tgcataccct
     3121 tatgatgtac cagactacgc tagggctaag agaggttcag gatctggagg cggatctggg
     3181 ggaggaaccg gtgctggaag tggtgagggc aggggatctc tccttacttg tggcgacgtg
     3241 gaggagaacc ccggccccat ggaattcggc ctgagctggc tgttcctggt ggccatcctg
     3301 aagggcgtgc agtgcagcag agacatcctg ctgacccaga cccctctgag cctgcctgtg
     3361 tctctgggcg atcaggccag catcagctgc agatccagcc agagcctggt gcaccggaac
     3421 ggcaacacct acctgcactg gtatctgcag aagcccggcc agagccccaa gctgctgatc
     3481 cacaaggtgt ccaaccggtt cagcggcgtg cccgacagat tttctggcag cggctccggc
     3541 accgacttca ccctgaagat cagccgggtg gaagccgagg acctgggcgt gtacttctgc
     3601 agccagtcca cccacgtgcc ccccctgaca tttggcgccg gaacaaagct ggaactgaag
     3661 gggggaggcg gatctggcgg cggaggaagt ggcggagggg gatctgaagt gaagctgcag
     3721 cagtccggcc ccagcctggt ggaacctggc gcctctgtga tgatctcctg caaggccagc
     3781 ggcagctcct tcaccggcta caacatgaac tgggtgcgcc agaacatcgg caagagcctg
     3841 gaatggatcg gcgccatcga cccctactac ggcggcacca gctacaacca gaagttcaag
     3901 ggcagagcca ccctgaccgt ggacaagagc agcagcaccg cctacatgca cctgaagtcc
     3961 ctgaccagcg aggacagcgc cgtgtactac tgcgtgtccg gcatgaagta ctggggccag
     4021 ggcacaagcg tgaccgtgtc tagcgccaag accacccccc ctagcgtgta cggaagagtg
     4081 acagtgtcct ctgccgagcc caagagctgc gacaagaccc acacctgtcc cccttgtcct
     4141 gcccctcctg ttgcaggccc ttccgtgttc ctgttccccc caaagcccaa ggacacactg
     4201 atgatcagca gaacccccga agtgacctgc gtggtggtgg acgtgtccca cgaggaccca
     4261 gaagtgaagt tcaattggta cgtggacggc gtggaagtgc acaacgccaa gacaaagccc
     4321 agagaggaac agtaccaaag cacctaccgg gtggtgtccg tgctgaccgt gctgcatcag
     4381 gattggctga acggcaaaga gtacaagtgc aaagtgtcca acaaggccct gcctgccccc
     4441 atcgagaaaa ccatcagcaa ggccaagggc cagccccgcg aaccccaggt gtacacactg
     4501 ccccctagca gggacgagct gaccaagaac caggtgtccc tgacatgcct cgtgaagggc
     4561 ttctacccct ccgatatcgc cgtggaatgg gagagcaacg gccagcccga gaacaactac
     4621 aagacaaccc ctcccgtgct ggacagcgac ggctcattct tcctgtacag caagctgaca
     4681 gtggataagt cccggtggca gcagggcaac gtgttcagct gctccgtgat gcacgaggcc
     4741 ctgcacaacc actacaccca gaaaagcctg tccctgagcc ccggcaagaa ggaccccaaa
     4801 gcggccgcaa ttgaagttat gtatcctcct ccttacctag acaatgagaa gagcaatgga
     4861 accattatcc atgtgaaagg gaaacacctt tgtccaagtc ccctatttcc cggaccttct
     4921 aagccctttt gggtgctggt ggtggttggg ggagtcctgg cttgctatag cttgctagta
     4981 acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac
     5041 tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc
     5101 ccaccacgcg acttcgcagc ctatcgctcc agagtgaagt tcagcaggag cgcagacgcc
     5161 cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag
     5221 gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga
     5281 aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc
     5341 tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac
     5401 cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc
     5461 cctcgccggg ccaagcggtc cggatccgga gccaccaact tcagcctgct gaagcaggcc
     5521 ggcgacgtgg aggagaaccc cggccccgcg gtgtaccagc tgagagactc taaatccagt
     5581 gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag
     5641 gattctgatg tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag
     5701 agcaacagtg ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac
     5761 aacagcatta ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc
     5821 ttcgcaggct gtttccttgc ttcaggaatg gccaggggat cctctagagt cgacctgcag
     5881 gcatgcaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     5941 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     6001 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6061 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6121 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     6181 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6241 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6301 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6361 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6421 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6481 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6541 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6601 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6661 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6721 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6781 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6841 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaa
//
