LOCUS       sequence-256362-        7412 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 7412)
  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 7412)
  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-ASCL2
FEATURES             Location/Qualifiers
     source          1..7412
                     /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    2389..2977
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2442..2462)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2860..2871)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3057..3191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3081..3100)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3135..3154
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3969..4031
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4161..4178)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4161..4177)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4170..4192)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4319..4774
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4406..4425)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4616..4637
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4800..4904
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4905..4973,4974..5765)
                     /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(5123..5142)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5936..6524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6425..6444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6678..6695
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6812..6833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6848..6853,6854..6871,6872..6878)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6886..6902
                     /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     6891..6913
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6910..6926
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6910..6926
                     /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 gcggcatgga cggcggcaca ctgcccaggt
     1801 ccgcgccccc tgcgcccccc gtccctgtcg gctgcgctgc ccggcggaga cccgcgtccc
     1861 cggaactgtt gcgctgcagc cggcggcggc gaccggccac cgcagagacc ggaggcggcg
     1921 cagcggccgt agcgcggcgc aatgagcgcg agcgcaaccg cgtgaagctg gtgaacttgg
     1981 gcttccaggc gctgcggcag cacgtgccgc acggcggcgc cagcaagaag ctgagcaagg
     2041 tggagacgct gcgctcagcc gtggagtaca tccgcgcgct gcagcgcctg ctggccgagc
     2101 acgacgccgt gcgcaacgcg ctggcgggag ggctgaggcc gcaggccgtg cggccgtctg
     2161 cgccccgcgg gccgccaggg accaccccgg tcgccgcctc gccctcccgc gcttcttcgt
     2221 ccccgggccg cgggggcagc tcggagcccg gctccccgcg ttccgcctac tcgtcggacg
     2281 acagcggctg cgaaggcgcg ctgagtcctg cggagcgcga gctactcgac ttctccagct
     2341 ggttaggggg ctactgagcg ccctcgagct agagttcgcg tctggaacaa tcaacctctg
     2401 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     2461 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     2521 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     2581 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     2641 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     2701 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     2761 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     2821 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     2881 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     2941 cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattaattc tggctaagca
     3001 cttcgtggcc gtcgatcgtt taaagggagg tagtgagtcg agatatcgat actagtgatc
     3061 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     3121 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     3181 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     3241 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc
     3301 tagagggaca gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg
     3361 gggcagcagc gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag
     3421 ccggcagcgt gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg
     3481 aacgcttctc gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaagccata
     3541 ccaatggagc tcacggggac agcccccccc caaagccccc agggatgtaa ttacgtccct
     3601 cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg cgctcccccc
     3661 gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg gcacgggatc
     3721 gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg gggatacggg
     3781 gaaaaagaat tcgataaaag ttttgttact ttatagaaga aattttgagt ttttgttttt
     3841 tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag tatgtaagtg
     3901 taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat atacagaccg
     3961 ataaaacaca tgcgtcaatt ttacacatga ttatctttaa cgtacgtcac aatatgatta
     4021 tctttctagg gttaatctag ctgcgtgttc tgcagcgtgt cgagcatctt catctgctcc
     4081 atcacgctgt aaaacacatt tgcaccgcga gtctgcccgt cctccacggg ttcaaaaacg
     4141 tgaatgaacg aggcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4201 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     4261 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     4321 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4381 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4441 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4501 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4561 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4621 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4681 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4741 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4801 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4861 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4921 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4981 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5041 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5101 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5161 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5221 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5281 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5341 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5401 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5461 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5521 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5581 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5641 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5701 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5761 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5821 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5881 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5941 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6001 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6061 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     6121 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6181 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6241 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6301 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6361 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6421 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6481 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6541 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6601 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6661 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6721 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6781 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6841 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6901 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcccgc cgggtaactc
     6961 acggggtatc catgtccatt tctgcggcat ccagccagga tacccgtcct cgctgacgta
     7021 atatcccagc gccgcaccgc tgtcattaat ctgcacaccg gcacggcagt tccggctgtc
     7081 gccggtattg ttcgggttgc tgatgcgctt cgggctgacc atccggaact gtgtccggaa
     7141 aagccgcgac gaactggtat cccaggtggc ctgaacgaac agttcaccgt taaaggcgtg
     7201 catggccaca ccttcccgaa tcatcatggt aaacgtgcgt tttcgctcaa cgtcaatgca
     7261 gcagcagtca tcctcggcaa actctttcca tgccgcttca acctcgcggg aaaaggcacg
     7321 ggcttcttcc tccccgatgc ccagatagcg ccagcttggg cgatgactga gccggaaaaa
     7381 agacccgacg atatgatcct gatgcagcta ga
//
