LOCUS       sequence-412268-        5962 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 5962)
  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 5962)
  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-704_CD19-28z_CAR_tNGFR
FEATURES             Location/Qualifiers
     source          1..5962
                     /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             3796..3825
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4558..4614
                     /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(4972..4988)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4972..4988)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4985..5007)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4996..5012
                     /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(5020..5026,5027..5044,5045..5050))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5065..5086
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5203..5220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5374..5962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5454..5473)
                     /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 gagaaccacg 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 gccctttcga ctcgcgcgtt tcggtgatga cggtgaaaac
     1261 ctctgacaca tgcagctccc ggagatggtc 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 ccccatgctt
     3001 ctcctggtga caagccttct gctctgtgag ttaccacacc cagcattcct cctgatccca
     3061 gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc
     3121 atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca
     3181 gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca
     3241 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa
     3301 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg
     3361 gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag
     3421 ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca
     3481 cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc
     3541 tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa
     3601 accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag
     3661 agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt
     3721 gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca
     3781 gtcaccgtct cctcagagca aaagctcatt tctgaagagg acttgttcgt gccggtcttc
     3841 ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc
     3901 gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg
     3961 cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact
     4021 tgtggggtcc ttctcctgtc actggttatc accctttact gcaaccacag gaacaggagt
     4081 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc
     4141 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga
     4201 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat
     4261 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg
     4321 gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa
     4381 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg
     4441 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac
     4501 gacgcccttc acatgcaggc cctgccccct cgccgggcca agcggtccgg atccggagcc
     4561 accaacttca gcctgctgaa gcaggccggc gacgtggagg agaaccccgg ccccgcggtg
     4621 taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat
     4681 tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg
     4741 ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct
     4801 gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc
     4861 agcccaggta agggcagctt tggtgccttc gcaggctgtt tccttgcttc aggaatggcc
     4921 aggggatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct
     4981 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     5041 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5101 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5161 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5221 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5281 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5341 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5401 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5461 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5521 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5581 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5641 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5701 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5761 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5821 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5881 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5941 gcgcagaaaa aaaggatctc aa
//
