LOCUS       sequence-256348-        6842 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for constitutive expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6842)
  AUTHORS   Fischer AD, Veronese-Paniagua DA, Swaminathan S, Kashima H, Rubin 
            DC, Madison BB
  TITLE     The oncogenic function of PLAGL2 is mediated via ASCL2 and IGF2 and 
            a Wnt-independent mechanism in colorectal cancer.
  JOURNAL   Am J Physiol Gastrointest Liver Physiol. 2023 Jun 13. doi: 
            10.1152/ajpgi.00058.2022.
  PUBMED    37310750
REFERENCE   2  (bases 1 to 6842)
  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: "Am J 
            Physiol Gastrointest Liver Physiol. 2023 Jun 13. doi: 
            10.1152/ajpgi.00058.2022."
COMMENT     Sequence Label: BII-ChBtW
FEATURES             Location/Qualifiers
     source          1..6842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(308..324)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(308..324)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1257..1266
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1263..1658
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1677..1730
                     /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             1740..1763
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1819..2407
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1872..1892)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2290..2301)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2487..2621
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2511..2530)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2565..2584
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3399..3461
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3591..3608)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3591..3607)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3600..3622)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3749..4204
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3836..3855)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4046..4067
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4230..4334
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4335..4403,4404..5195)
                     /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(4553..4572)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5366..5954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5855..5874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6108..6125
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6242..6263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6278..6283,6284..6301,6302..6308)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6316..6332
                     /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     6321..6343
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6340..6356
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6340..6356
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat ccactagttc tagagggaca gccccccccc aaagccccca gggatgtaat
      361 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
      421 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
      481 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
      541 ggatacgggg aaaaggcctc caaggcctgc aggagtcaat gggaaaaacc cattggagcc
      601 aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag
      661 ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc
      721 caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt
      781 actgacatgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt
      841 caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga
      901 ctttccattg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc
      961 ccattattgg cacatacata aggtcaatag gggtgactag tggagaagag catgcttgag
     1021 ggctgagtgc ccctcagtgg gcagagagca catggcccac agtccctgag aagttggggg
     1081 gaggggtggg caattgaact ggtgcctaga gaaggtgggg cttgggtaaa ctgggaaagt
     1141 gatgtggtgt actggctcca cctttttccc cagggtgggg gagaaccata tataagtgca
     1201 gtagtctctg tgaacattcg gtttagtgaa cctgcaggtc ggccgccacg accggtgcca
     1261 ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     1321 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     1381 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     1441 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     1501 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     1561 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     1621 ttcgtgaatt gctgccctct ggttatgtgt gggagggcgc tagccttggc tccggcgagg
     1681 gcaggggaag tcttctaaca tgcggggacg tggaggaaaa tcccggccca ctcggcatgg
     1741 actacaagga cgacgatgac aagctcgata gcggcatgag acggatagct cgtctcagct
     1801 agagttcgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     1861 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     1921 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     1981 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     2041 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     2101 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     2161 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     2221 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     2281 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     2341 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     2401 tccccgcctg gaattaattc tggctaagca cttcgtggcc gtcgatcgtt taaagggagg
     2461 tagtgagtcg agatatcgat actagtgatc cagacatgat aagatacatt gatgagtttg
     2521 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     2581 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     2641 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     2701 acaaatgtgg tatggctgat tatgatcctc tagagggaca gccccccccc aaagccccca
     2761 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     2821 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     2881 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     2941 acacctgggg ggatacgggg aaaagccata ccaatggagc tcacggggac agcccccccc
     3001 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
     3061 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
     3121 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
     3181 tgagcctgca gacacctggg gggatacggg gaaaaagaat tcgataaaag ttttgttact
     3241 ttatagaaga aattttgagt ttttgttttt tttaataaat aaataaacat aaataaattg
     3301 tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa
     3361 attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacacatga
     3421 ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag ctgcgtgttc
     3481 tgcagcgtgt cgagcatctt catctgctcc atcacgctgt aaaacacatt tgcaccgcga
     3541 gtctgcccgt cctccacggg ttcaaaaacg tgaatgaacg aggcgcgctc actggccgtc
     3601 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     3661 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     3721 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     3781 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     3841 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     3901 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3961 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4021 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4081 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4141 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     4201 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4261 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4321 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4381 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4441 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4501 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4561 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4621 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4681 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4741 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4801 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4861 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4921 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4981 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     5041 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     5101 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     5161 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5221 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5281 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5341 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5401 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5461 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     5521 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5581 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5641 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5701 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5761 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5821 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5881 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5941 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     6001 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     6061 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     6121 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     6181 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     6241 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     6301 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     6361 attacgccaa gcgcgcccgc cgggtaactc acggggtatc catgtccatt tctgcggcat
     6421 ccagccagga tacccgtcct cgctgacgta atatcccagc gccgcaccgc tgtcattaat
     6481 ctgcacaccg gcacggcagt tccggctgtc gccggtattg ttcgggttgc tgatgcgctt
     6541 cgggctgacc atccggaact gtgtccggaa aagccgcgac gaactggtat cccaggtggc
     6601 ctgaacgaac agttcaccgt taaaggcgtg catggccaca ccttcccgaa tcatcatggt
     6661 aaacgtgcgt tttcgctcaa cgtcaatgca gcagcagtca tcctcggcaa actctttcca
     6721 tgccgcttca acctcgcggg aaaaggcacg ggcttcttcc tccccgatgc ccagatagcg
     6781 ccagcttggg cgatgactga gccggaaaaa agacccgacg atatgatcct gatgcagcta
     6841 ga
//
