LOCUS       sequence-344455-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PiggyBack transposon expressing a fusion of a tandem minigene 
            antigens in KRAS, BRAF and CMV fused to an endosomal targeting 
            sequence and translationally linked to human truncated CD19.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7919)
  AUTHORS   Veatch JR, Singhi N, Srivastava S, Szeto JL, Jesernig B, Stull SM, 
            Fitzgibbon M, Sarvothama M, Yechan-Gunja S, James SE, Riddell SR
  TITLE     A therapeutic cancer vaccine delivers antigens and adjuvants to 
            lymphoid tissues using genetically modified T cells.
  JOURNAL   J Clin Invest. 2021 Aug 16;131(16):e144195. doi: 10.1172/JCI144195.
  PUBMED    34396986
REFERENCE   2  (bases 1 to 7919)
  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"; journalName: "J Clin 
            Invest."; date: "2021-08-16"; volume: "131(16):e144195. doi"; pages:
            " 10.1172/JCI144195"
COMMENT     Sequence Label: PB713B-pCMV-MITD-NeoMini-T2A-tCD19-WPRE
FEATURES             Location/Qualifiers
     source          1..7919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..81)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(235..823)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(315..334)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(994..1785,1786..1854))
                     /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     1617..1636
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1855..1959)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1985..2440)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2122..2143)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2334..2353
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2567..2589
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2581..2598
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2582..2598
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   2728..2790
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        3232..3611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3612..3815
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3766..3786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3860..3879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3860..3878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4678..4731
                     /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"
     misc_feature    5757..6345
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5810..5830)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6228..6239)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6369..6593
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7227..7261
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7752..7768)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7752..7768)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7765..7787)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7776..7792
                     /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(7800..7806,7807..7824,7825..7830))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7845..7866
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
       61 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      121 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      181 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      241 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      301 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      361 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      421 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      481 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      541 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      601 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      661 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      721 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      781 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
      841 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
      901 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
      961 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1021 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1081 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1141 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1201 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1261 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1321 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1381 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1441 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1501 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1561 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1621 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1681 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1741 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     1801 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     1861 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     1921 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     1981 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2041 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2101 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2161 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2221 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2281 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     2341 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     2401 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     2461 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
     2521 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
     2581 tgtaaaacga cggccagtga gcgcgcctcg ttcattcacg tttttgaacc cgtggaggac
     2641 gggcagactc gcggtgcaaa tgtgttttac agcgtgatgg agcagatgaa gatgctcgac
     2701 acgctgcaga acacgcagct agattaaccc tagaaagata atcatattgt gacgtacgtt
     2761 aaagataatc atgcgtaaaa ttgacgcatg tgttttatcg gtctgtatat cgaggtttat
     2821 ttattaattt gaatagatat taagttttat tatatttaca cttacatact aataataaat
     2881 tcaacaaaca atttatttat gtttatttat ttattaaaaa aaacaaaaac tcaaaatttc
     2941 ttctataaag taacaaaact tttatgaggg acagcccccc cccaaagccc ccagggatgt
     3001 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc
     3061 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg
     3121 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg
     3181 gggggatacg gggaaaaggc ctccacggcc actagttgaa agaccccacg cgacattgat
     3241 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
     3301 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     3361 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     3421 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     3481 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     3541 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     3601 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     3661 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     3721 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     3781 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     3841 cttactggct tatcgaaatt aatacgactc actataggga gacccaagct ggctagctcc
     3901 accatgagag tgacagcccc tagaacactg ctcctgctgc tgtggggagc tgtggccctt
     3961 acagaaacat gggccggcag ctctggcagc cacgaagatc tgacagtgaa gatcggcgac
     4021 ttcggcctgg ccaccgagaa atctcgttgg agcggcagcc accagttcga gcagctgtct
     4081 ggctctatcc tgagcagcgg ctctagcgtc gtgggcagca aggatatgag cacctgggtg
     4141 ttcggcaccg agagatggga caacctgatc tactatgccc tcggcggcag cagcggatct
     4201 tctcaggctg gaatcctggc cagaaacctg gtgcctatgg tggccacagt gcagggccag
     4261 aatctgaagt ctagcggcag cggaagcccc aaggccaaca aagagattct ggacgaggcc
     4321 tacgtgatgg cttatgtgat ggccggcgtg ggctctccct atgtgtctag actgctgggc
     4381 agctccggaa gcagcatgac cgagtataag ctggtggtcg tgggcgccgt tggcgtggga
     4441 aaatctgccc tgacaatcca gctgatccag tccagcggat cctccacagg caccggtatt
     4501 gtaggcatcg tggcaggctt ggctgttctg gccgttgtcg ttatcggcgc agttgtggcc
     4561 gccgtcatgt gccggcggaa atcctccggc ggcaaaggcg gttcttactc ccaggctgct
     4621 tgttcagatt ctgcccaggg gagtgacgtt tccttgaccg ctctcgaggg cggcggagag
     4681 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atccaggccc taggatgcca
     4741 cctccaagac tcctcttctt cctcctcttc ctgacaccaa tggaagtcag gcctgaggaa
     4801 cctctagtgg tgaaggtgga agagggagat aacgctgtgt tacagtgcct caagggaacc
     4861 tcagatggac ccactcagca gctgacctgg tctcgggagt ctccgcttaa acccttcctg
     4921 aaactcagcc ttggactgcc aggtctggga atccacatga ggccactggc tatctggctg
     4981 ttcatcttca acgtctctca acagatggga ggcttctacc tgtgtcagcc tggaccacct
     5041 tctgagaagg catggcagcc tggttggaca gtcaatgtgg agggttctgg tgagctgttc
     5101 cggtggaatg tttcggacct aggtggactg ggatgtggtc tgaagaacag gtcctcagag
     5161 ggacctagct ctccttccgg gaagctcatg agccccaagc tgtatgtgtg ggccaaagac
     5221 cgccctgaga tctgggaggg agagcctccg tgtgtcccac cgagggacag cctgaaccag
     5281 agcctcagcc aggacctcac catggcccct ggctccacac tctggctgtc ctgtggggta
     5341 ccccctgact ctgtgtccag gggccccctc tcctggaccc atgtgcaccc caaggggcct
     5401 aagtcattgc tgagcctaga gctgaaggac gatcgccctg ccagagatat gtgggtaatg
     5461 gagacgggtc tgttgttgcc ccgggccaca gctcaagacg ctggaaagta ttattgtcac
     5521 cgtggcaacc tgaccatgtc attccacctg gagatcactg ctcggccagt actatggcac
     5581 tggctgctga ggactggtgg ctggaaggtc tcagctgtga ctttggctta tctgatcttc
     5641 tgcctgtgtt cccttgtggg cattcttcat cttcaaagag ccctggtcct gaggaggaaa
     5701 agatgagcgg ccgctctaga cccgggctgc aggaattcga tatcaagctt atcgataatc
     5761 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     5821 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     5881 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     5941 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     6001 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     6061 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     6121 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     6181 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     6241 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     6301 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac
     6361 tagcccgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6421 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6481 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     6541 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     6601 aggtcaacca ggttgcaggg tttcctgtcc tcacaggaac gaagtcccta aagaaacagt
     6661 ggcagccagg tttagccccg gaattgactg gattcctttt ttagggccca ttggtatggc
     6721 tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag
     6781 gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg
     6841 ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag
     6901 cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc ctgatatcta
     6961 taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata
     7021 attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact
     7081 cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg
     7141 ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag
     7201 aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg
     7261 gttaatctag ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc
     7321 aagctggcgc tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga
     7381 agcggcatgg aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg
     7441 cacgtttacc atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt
     7501 cgttcaggcc acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt
     7561 cagcccgaag cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg
     7621 tgtgcagatt aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc
     7681 tggctggatg ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcttg
     7741 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     7801 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     7861 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagct
//
