LOCUS       sequence-412295-        6963 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 6963)
  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 6963)
  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-805_HA-GD2-28z_CAR_BATF-RFP
FEATURES             Location/Qualifiers
     source          1..6963
                     /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             2545..3249
                     /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(2687..2705)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2955..2974
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3274..3327
                     /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             5560..5616
                     /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(5973..5989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5973..5989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5986..6008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5997..6013
                     /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(6021..6027,6028..6045,6046..6051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6066..6087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6204..6221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6375..6963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6455..6474)
                     /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 gtggtagtgg agggcagtgc accaactatg cgctgctgaa actggcgggc
     2521 gatgtggaaa gcaacccggg cccggtgagc aagggcgagg aggataacat ggccatcatc
     2581 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     2641 atcgagggtg agggagaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     2701 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     2761 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     2821 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     2881 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     2941 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     3001 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     3061 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     3121 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     3181 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     3241 ctgtacaaga gggctaagag aggttcagga tctgagggca ggggatctct ccttacttgt
     3301 ggcgacgtgg aggagaaccc cggccccatg gaattcggcc tgagctggct gttcctggtg
     3361 gccatcctga agggcgtgca gtgcagcaga gacatcctgc tgacccagac ccctctgagc
     3421 ctgcctgtgt ctctgggcga tcaggccagc atcagctgca gatccagcca gagcctggtg
     3481 caccggaacg gcaacaccta cctgcactgg tatctgcaga agcccggcca gagccccaag
     3541 ctgctgatcc acaaggtgtc caaccggttc agcggcgtgc ccgacagatt ttctggcagc
     3601 ggctccggca ccgacttcac cctgaagatc agccgggtgg aagccgagga cctgggcgtg
     3661 tacttctgca gccagtccac ccacgtgccc cccctgacat ttggcgccgg aacaaagctg
     3721 gaactgaagg ggggaggcgg atctggcggc ggaggaagtg gcggaggggg atctgaagtg
     3781 aagctgcagc agtccggccc cagcctggtg gaacctggcg cctctgtgat gatctcctgc
     3841 aaggccagcg gcagctcctt caccggctac aacatgaact gggtgcgcca gaacatcggc
     3901 aagagcctgg aatggatcgg cgccatcgac ccctactacg gcggcaccag ctacaaccag
     3961 aagttcaagg gcagagccac cctgaccgtg gacaagagca gcagcaccgc ctacatgcac
     4021 ctgaagtccc tgaccagcga ggacagcgcc gtgtactact gcgtgtccgg catgaagtac
     4081 tggggccagg gcacaagcgt gaccgtgtct agcgccaaga ccaccccccc tagcgtgtac
     4141 ggaagagtga cagtgtcctc tgccgagccc aagagctgcg acaagaccca cacctgtccc
     4201 ccttgtcctg cccctcctgt tgcaggccct tccgtgttcc tgttcccccc aaagcccaag
     4261 gacacactga tgatcagcag aacccccgaa gtgacctgcg tggtggtgga cgtgtcccac
     4321 gaggacccag aagtgaagtt caattggtac gtggacggcg tggaagtgca caacgccaag
     4381 acaaagccca gagaggaaca gtaccaaagc acctaccggg tggtgtccgt gctgaccgtg
     4441 ctgcatcagg attggctgaa cggcaaagag tacaagtgca aagtgtccaa caaggccctg
     4501 cctgccccca tcgagaaaac catcagcaag gccaagggcc agccccgcga accccaggtg
     4561 tacacactgc cccctagcag ggacgagctg accaagaacc aggtgtccct gacatgcctc
     4621 gtgaagggct tctacccctc cgatatcgcc gtggaatggg agagcaacgg ccagcccgag
     4681 aacaactaca agacaacccc tcccgtgctg gacagcgacg gctcattctt cctgtacagc
     4741 aagctgacag tggataagtc ccggtggcag cagggcaacg tgttcagctg ctccgtgatg
     4801 cacgaggccc tgcacaacca ctacacccag aaaagcctgt ccctgagccc cggcaagaag
     4861 gaccccaaag cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag
     4921 agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc
     4981 ggaccttcta agcccttttg ggtgctggtg gtggttgggg gagtcctggc ttgctatagc
     5041 ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg
     5101 cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag
     5161 ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc
     5221 gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga
     5281 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga
     5341 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg
     5401 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat
     5461 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag
     5521 gccctgcccc ctcgccgggc caagcggtcc ggatccggag ccaccaactt cagcctgctg
     5581 aagcaggccg gcgacgtgga ggagaacccc ggccccgcgg tgtaccagct gagagactct
     5641 aaatccagtg acaagtctgt ctgcctattc accgattttg attctcaaac aaatgtgtca
     5701 caaagtaagg attctgatgt gtatatcaca gacaaaactg tgctagacat gaggtctatg
     5761 gacttcaaga gcaacagtgc tgtggcctgg agcaacaaat ctgactttgc atgtgcaaac
     5821 gccttcaaca acagcattat tccagaagac accttcttcc ccagcccagg taagggcagc
     5881 tttggtgcct tcgcaggctg tttccttgct tcaggaatgg ccaggggatc ctctagatcg
     5941 acctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     6001 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     6061 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6121 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6181 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6241 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     6301 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6361 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6421 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6481 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6541 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6601 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6661 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6721 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6781 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6841 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6901 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6961 caa
//
