LOCUS       sequence-412261-        6631 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 6631)
  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 6631)
  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-671_HA-GD2-28z_CAR_tNGFR
FEATURES             Location/Qualifiers
     source          1..6631
                     /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             2941..2994
                     /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             5227..5283
                     /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(5641..5657)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5641..5657)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5654..5676)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5665..5681
                     /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(5689..5695,5696..5713,5714..5719))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5734..5755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5872..5889)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6043..6631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6123..6142)
                     /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 cggcggttct
     1981 ggtggtcagt gcaccaacta tgcgctgctg aaactggcgg gcgatgtgga aagcaacccg
     2041 ggcccgatgg gcgctggtgc aactggacgg gccatggatg gaccacggct tttgctcctg
     2101 ttgttgttgg gcgtcagcct tggaggggct aaagaggcct gcccgaccgg tttgtacaca
     2161 cactccggcg agtgttgtaa ggcgtgtaat cttggcgaag gggtcgccca accgtgtggc
     2221 gccaaccaga cggtttgtga gccttgcttg gatagcgtca cgttttccga cgtcgtttca
     2281 gccaccgaac catgcaaacc ttgtacggaa tgcgtgggtc tgcagagcat gagcgcaccg
     2341 tgcgtagagg cggacgatgc ggtatgccga tgtgcctacg gctactacca agacgagacg
     2401 acagggaggt gtgaggcatg cagagtatgt gaggccgggt ctggactcgt attttcctgt
     2461 caagacaaac aaaacacagt atgtgaggag tgcccggatg ggacttattc cgatgaagcg
     2521 aatcatgtgg acccgtgctt gccatgtacg gtctgtgagg ataccgaaag acagctccga
     2581 gaatgcaccc gctgggctga cgcggaatgc gaagaaatcc caggtcgatg gatcacccgc
     2641 tcaacccccc ccgaagggtc agattctacg gcgccctcta ctcaggagcc agaagctcca
     2701 cccgaacaag acctgatcgc ttctacggtg gcaggtgtcg ttacaacagt catgggttcc
     2761 tcacaacctg tggttacgcg ggggacaact gacaatttga ttccagtata ttgtagcatt
     2821 ctggccgcgg tcgtggtagg gcttgtcgcg tatatagcgt tcaaaaggtg gaacagcagg
     2881 gctaagagag gttcaggatc tggggggggc tcgggaggtg gaaccggtgc tggaagtggt
     2941 gagggcaggg gatctctcct tacttgtggc gacgtggagg agaaccccgg ccccatggaa
     3001 ttcggcctga gctggctgtt cctggtggcc atcctgaagg gcgtgcagtg cagcagagac
     3061 atcctgctga cccagacccc tctgagcctg cctgtgtctc tgggcgatca ggccagcatc
     3121 agctgcagat ccagccagag cctggtgcac cggaacggca acacctacct gcactggtat
     3181 ctgcagaagc ccggccagag ccccaagctg ctgatccaca aggtgtccaa ccggttcagc
     3241 ggcgtgcccg acagattttc tggcagcggc tccggcaccg acttcaccct gaagatcagc
     3301 cgggtggaag ccgaggacct gggcgtgtac ttctgcagcc agtccaccca cgtgcccccc
     3361 ctgacatttg gcgccggaac aaagctggaa ctgaaggggg gaggcggatc tggcggcgga
     3421 ggaagtggcg gagggggatc tgaagtgaag ctgcagcagt ccggccccag cctggtggaa
     3481 cctggcgcct ctgtgatgat ctcctgcaag gccagcggca gctccttcac cggctacaac
     3541 atgaactggg tgcgccagaa catcggcaag agcctggaat ggatcggcgc catcgacccc
     3601 tactacggcg gcaccagcta caaccagaag ttcaagggca gagccaccct gaccgtggac
     3661 aagagcagca gcaccgccta catgcacctg aagtccctga ccagcgagga cagcgccgtg
     3721 tactactgcg tgtccggcat gaagtactgg ggccagggca caagcgtgac cgtgtctagc
     3781 gccaagacca ccccccctag cgtgtacgga agagtgacag tgtcctctgc cgagcccaag
     3841 agctgcgaca agacccacac ctgtccccct tgtcctgccc ctcctgttgc aggcccttcc
     3901 gtgttcctgt tccccccaaa gcccaaggac acactgatga tcagcagaac ccccgaagtg
     3961 acctgcgtgg tggtggacgt gtcccacgag gacccagaag tgaagttcaa ttggtacgtg
     4021 gacggcgtgg aagtgcacaa cgccaagaca aagcccagag aggaacagta ccaaagcacc
     4081 taccgggtgg tgtccgtgct gaccgtgctg catcaggatt ggctgaacgg caaagagtac
     4141 aagtgcaaag tgtccaacaa ggccctgcct gcccccatcg agaaaaccat cagcaaggcc
     4201 aagggccagc cccgcgaacc ccaggtgtac acactgcccc ctagcaggga cgagctgacc
     4261 aagaaccagg tgtccctgac atgcctcgtg aagggcttct acccctccga tatcgccgtg
     4321 gaatgggaga gcaacggcca gcccgagaac aactacaaga caacccctcc cgtgctggac
     4381 agcgacggct cattcttcct gtacagcaag ctgacagtgg ataagtcccg gtggcagcag
     4441 ggcaacgtgt tcagctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaaa
     4501 agcctgtccc tgagccccgg caagaaggac cccaaagcgg ccgcaattga agttatgtat
     4561 cctcctcctt acctagacaa tgagaagagc aatggaacca ttatccatgt gaaagggaaa
     4621 cacctttgtc caagtcccct atttcccgga ccttctaagc ccttttgggt gctggtggtg
     4681 gttgggggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg
     4741 gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc
     4801 cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat
     4861 cgctccagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac
     4921 cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga
     4981 cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg
     5041 tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc
     5101 gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag
     5161 gacacctacg acgcccttca catgcaggcc ctgccccctc gccgggccaa gcggtccgga
     5221 tccggagcca ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggc
     5281 cccgcggtgt accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc
     5341 gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac
     5401 aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc
     5461 aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc
     5521 ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg caggctgttt ccttgcttca
     5581 ggaatggcca ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg
     5641 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5701 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5761 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5821 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5881 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5941 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     6001 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     6061 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     6121 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     6181 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6241 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6301 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6361 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6421 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6481 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6541 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6601 gcagattacg cgcagaaaaa aaggatctca a
//
