LOCUS       sequence-412326-        5536 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 5536)
  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 5536)
  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-837_NY-ESO-1_TCR_tFAS
FEATURES             Location/Qualifiers
     source          1..5536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(1764..1820)
                     /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"
     CDS             complement(2778..2831)
                     /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             complement(3486..3545)
                     /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"
     primer_bind     complement(3911..3928)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3911..3927)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3920..3942)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4136..4155)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4255..4277
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4315..4333)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4401..4505
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4506..4574,4575..5366)
                     /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     complement(4724..4743)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg catgcctgca
     1021 ggtcgactct agaggatggc cattcctgaa gcaaggaaac agcctgcgaa ggcaccaaag
     1081 ctgcccttac ctgggctggg gaagaaggtg tcttctggaa taatgctgtt gttgaaggcg
     1141 tttgcacatg caaagtcaga tttgttgctc caggccacag cactgttgct cttgaagtcc
     1201 atagacctca tgtctagcac agttttgtct gtgatataca catcagaatc cttactttgt
     1261 gacacatttg tttgagaatc aaaatcggtg aataggcaga cagacttgtc actggattta
     1321 gagtctctca gctggtacac cgcagggtca gggttctgga tatacggatg aacaataagg
     1381 ctggttcctc ttccaaatgt aggtatgtag cttcctccgt acaggggcct cacagcacag
     1441 aggtaggtgg ctgagtcacc aggctgagaa gctgcaatgt ataaagtact acgtcctgat
     1501 gatttatcca gcgaggcatt aagtcttcca cttgtttgct ctctctgact tgactgaata
     1561 agcaacagag atgtgagacc tttcccaggg tcctgcctaa accactggag gttgtaaata
     1621 gcgctatcag tgaaactgca gttgagaacc aagttttctc cttctgggac actcagagct
     1681 gcaggaatct gcgtcacctc ctgtttgctg ctcacccatt gcagctgcag ccaaaggata
     1741 agcaggccca agagggtctc catggggccg gggttctcct ccacgtcgcc ggcctgcttc
     1801 agcaggctga agttggtggc tccggatccg gaccgcttgg cccggcctct ggaatccttt
     1861 ctcttgacca tagccatcag cacgagggca ctgaccagca cggcatacaa ggtggccttc
     1921 cctagcaaga tctcatagag gatggtggca gacaggaccc cttgctggta agactcggag
     1981 gtgaagccac agtctgctct accccaggcc tcggcgctga cgatctgggt gacgggtttg
     2041 gccctatcct gggtccactc gtcattctcc gagagcccgt agaactggac ttgacagcgg
     2101 aagtggttgc gggggttctg ccagaaggtg gccgagaccc tcaggcggct gctcaggcag
     2161 tatctggagt cattgagggc gggctgctcc ttgaggggct gcgggtctgt gctgacccca
     2221 ctgtgcacct ccttcccatt cacccaccag ctcagctcca cgtggtcggg gtagaagcct
     2281 gtggccaggc ataccagtgt ggccttttgg gtgtgggaga tctctgcttc tgatggctca
     2341 aacacagcga cctcgggtgg gaacacgttt ttcaggtcct ccagtacggt cagcctagag
     2401 ccttctccaa aaaacagctc cccggtgttc ccgacgtaac tgctggcaca gaagtacaca
     2461 gatgtctggg agggagcagc cgacagcagc ctgagcggga aatcctctgt ggttgatctg
     2521 gagacattgt agccattggg gacttctcct tggtcagtga taccagcacc aactgagtaa
     2581 tgaatcagcc tcagccccat gcctgggtct tgtcgatacc aggacatgta ttcatggttc
     2641 atatcctggg cacactgcag tgtcatgctc tgtcctgtct tcaggacctg gaattttggg
     2701 gtctgagtga caccagcatt cactggacct gcccacagga gagacaaggc tgcacagcac
     2761 aggaggccga tgctcatggg gccggggttc tcctccacgt cgccacaagt aaggagagat
     2821 cccctgccct caccacttcc agcaccggtt cctccgccag atccgccacc agatcctgaa
     2881 cctctcttag cccttttctg tacttccttt ctcttcaccc aaacaattag tggaattggc
     2941 aaaagaagaa gacaaagcca ccccaagtta gatctggatc cttcctcttt gcacttggtg
     3001 ttgctggtga gtgtgcattc cttgatgatt ccatgttcac atttggtgca agggtcacag
     3061 tgttcacata cagtagagtt acaaaaaaag tttggtttac atctgcactt ggtattctgg
     3121 gtccgggtgc agtttatttc cacttctaag ccatgtcctt catcacacaa tctacatctt
     3181 ctgcatttgg aagaaaaatg ggctttgtct gtgtactcct tcccttcttg gcagggcacg
     3241 cagtctggtt catccccatt gactgtgcag tccctagctt tcctttcacc tggaggacag
     3301 ggcttatggc agaattggcc atcatgatgc aggccttcca agttctgagt ctcaactgta
     3361 gtaacagtct tcctcaattc caatcccttg gagttgatgt cagtcacttg ggcattaaca
     3421 cttttggacg ataatctagc aacagacgta agaaccagag gtaggagggt ccagatgccc
     3481 agcatcgggc ccgggttgct ttccacatcg cccgccagtt tcagcagcgc atagttggtg
     3541 cactgtcccc cacttccacc accgccacta gtcccacccg atccggcagg gtcagggttc
     3601 tggatatctg tgggacaaga ggatcagggt taggacatga tctcatttcc ctctttgccc
     3661 caacccaggc tggagtccag atgccagtga tggacaaggg cggggctctg tgggcgtgcg
     3721 aatgcacgtg ctcatgcttt atacgggaaa tagcatctta gaaaccagct gctcgtgatg
     3781 gactgggact cagggacagg cacaagctat caatcttggc caagaggcca tgatttcagt
     3841 gaacgttcac ggccaggcct ggcctgccac tcaaggaaac ctgaaatccc cgggtaccga
     3901 gctcgaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     3961 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4021 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg tgaatggcgc ctgatgcggt
     4081 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4141 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4201 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4261 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4321 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4381 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     4441 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     4501 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4561 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4621 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4681 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4741 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4801 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4861 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4921 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4981 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5041 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5101 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5161 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5221 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5281 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5341 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     5401 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     5461 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5521 agatcaaagg atcttc
//
