LOCUS       sequence-412304-        5659 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 5659)
  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 5659)
  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-833_NY-ESO-1_TCR_FAS/CD28
FEATURES             Location/Qualifiers
     source          1..5659
                     /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             1992..2051
                     /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             2829..2882
                     /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             3840..3896
                     /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(4669..4685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4669..4685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4682..4704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4693..4709
                     /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(4717..4723,4724..4741,4742..4747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4762..4783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4900..4917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5071..5659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5151..5170)
                     /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 cattcaccat tcaggctgcg caactgttgg
     1501 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     1561 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     1621 gccagtgaat tcgagctcgg tacccgggga tttcaggttt ccttgagtgg caggccaggc
     1681 ctggccgtga acgttcactg aaatcatggc ctcttggcca agattgatag cttgtgcctg
     1741 tccctgagtc ccagtccatc acgagcagct ggtttctaag atgctatttc ccgtataaag
     1801 catgagcacg tgcattcgca cgcccacaga gccccgccct tgtccatcac tggcatctgg
     1861 actccagcct gggttggggc aaagagggaa atgagatcat gtcctaaccc tgatcctctt
     1921 gtcccacaga tatccagaac cctgaccctg ccggatcggg tgggactagt ggcggaggtg
     1981 ggagcggcgg acagtgcacc aactatgcgc tgctgaaact ggcgggcgat gtggaaagca
     2041 acccgggccc gatgctgggc atctggaccc tcctacctct ggttcttacg tctgttgcta
     2101 gattatcgtc caaaagtgtt aatgcccaag tgactgacat caactccaag ggattggaat
     2161 tgaggaagac tgttactaca gttgagactc agaacttgga aggcctgcat catgatggcc
     2221 aattctgcca taagccctgt cctccaggtg aaaggaaagc tagggactgc acagtcaatg
     2281 gggatgaacc agactgcgtg ccctgccaag aagggaagga gtacacagac aaagcccatt
     2341 tttcttccaa atgcagaaga tgtagattgt gtgatgaagg acatggctta gaagtggaaa
     2401 taaactgcac ccggacccag aataccaagt gcagatgtaa accaaacttt ttttgtaact
     2461 ctactgtatg tgaacactgt gacccttgca ccaaatgtga acatggaatc atcaaggaat
     2521 gcacactcac cagcaacacc aagtgcaaag aggaaggatc cagatctaac ttggggtggc
     2581 tttgtcttct tcttttgcca attccactaa ttgtttgggt gaagagaaag gaagtacaga
     2641 aaaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc
     2701 ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc
     2761 gctccagggc taagagaggt tcaggatctg gtggtgggtc tggcggagga accggtgctg
     2821 gaagtggtga gggcagggga tctctcctta cttgtggcga cgtggaggag aaccccggcc
     2881 ccatgagcat cggcctcctg tgctgtgcag ccttgtctct cctgtgggca ggtccagtga
     2941 atgctggtgt cactcagacc ccaaaattcc aggtcctgaa gacaggacag agcatgacac
     3001 tgcagtgtgc ccaggatatg aaccatgaat acatgtcctg gtatcgacaa gacccaggca
     3061 tggggctgag gctgattcat tactcagttg gtgctggtat cactgaccaa ggagaagtcc
     3121 ccaatggcta caatgtctcc agatcaacca cagaggattt cccgctcagg ctgctgtcgg
     3181 ctgctccctc ccagacatct gtgtacttct gtgccagcag ttacgtcggg aacaccgggg
     3241 agctgttttt tggagaaggc tctaggctga ccgtactgga ggacctgaaa aacgtgttcc
     3301 cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc caaaaggcca
     3361 cactggtatg cctggccaca ggcttctacc ccgaccacgt ggagctgagc tggtgggtga
     3421 atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag gagcagcccg
     3481 ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc accttctggc
     3541 agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg gagaatgacg
     3601 agtggaccca ggatagggcc aaacccgtca cccagatcgt cagcgccgag gcctggggta
     3661 gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct gccaccatcc
     3721 tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt gccctcgtgc
     3781 tgatggctat ggtcaagaga aaggattcca gaggccgggc caagcggtcc ggatccggag
     3841 ccaccaactt cagcctgctg aagcaggccg gcgacgtgga ggagaacccc ggccccatgg
     3901 agaccctctt gggcctgctt atcctttggc tgcagctgca atgggtgagc agcaaacagg
     3961 aggtgacgca gattcctgca gctctgagtg tcccagaagg agaaaacttg gttctcaact
     4021 gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac cctgggaaag
     4081 gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt ggaagactta
     4141 atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct tctcagcctg
     4201 gtgactcagc cacctacctc tgtgctgtga ggcccctgta cggaggaagc tacataccta
     4261 catttggaag aggaaccagc cttattgttc atccgtatat ccagaaccct gaccctgcgg
     4321 tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc accgattttg
     4381 attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca gacaaaactg
     4441 tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg agcaacaaat
     4501 ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac accttcttcc
     4561 ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct tcaggaatgg
     4621 ccatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt
     4681 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4741 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4801 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4861 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4921 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4981 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5041 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5101 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5161 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5221 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5281 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5341 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5401 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5461 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5521 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5581 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5641 cagaaaaaaa ggatctcaa
//
