LOCUS       sequence-267632-        7413 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 7413)
  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 7413)
  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..7413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(269..285)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(269..285)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1218..1227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1224..1619
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1638..1691
                     /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             1701..1724
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2350..2938
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2403..2423)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2821..2832)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3018..3152
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3042..3061)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3096..3115
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3931..3993
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4123..4140)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4123..4139)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4132..4154)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4281..4736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4368..4387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4578..4599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4762..4866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4867..4935,4936..5727)
                     /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(5085..5104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5898..6486
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6387..6406
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6640..6657
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6774..6795
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6810..6815,6816..6833,6834..6840)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6848..6864
                     /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     6853..6875
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6872..6888
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6872..6888
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7379..7413
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg
       61 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac
      121 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac
      181 ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa
      241 cttattatat atatattttc ttgttataga tccactagtt ctagagggac agcccccccc
      301 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
      361 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
      421 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
      481 tgagcctgca gacacctggg gggatacggg gaaaaggcct ccaaggcctg caggagtcaa
      541 tgggaaaaac ccattggagc caagtacact gactcaatag ggactttcca ttgggttttg
      601 cccagtacat aaggtcaata gggggtgagt caacaggaaa gtcccattgg agccaagtac
      661 attgagtcaa tagggacttt ccaatgggtt ttgcccagta cataaggtca atgggaggta
      721 agccaatggg tttttcccat tactgacatg tatactgagt cattagggac tttccaatgg
      781 gttttgccca gtacataagg tcaatagggg tgaatcaaca ggaaagtccc attggagcca
      841 agtacactga gtcaataggg actttccatt gggttttgcc cagtacaaaa ggtcaatagg
      901 gggtgagtca atgggttttt cccattattg gcacatacat aaggtcaata ggggtgacta
      961 gtggagaaga gcatgcttga gggctgagtg cccctcagtg ggcagagagc acatggccca
     1021 cagtccctga gaagttgggg ggaggggtgg gcaattgaac tggtgcctag agaaggtggg
     1081 gcttgggtaa actgggaaag tgatgtggtg tactggctcc acctttttcc ccagggtggg
     1141 ggagaaccat atataagtgc agtagtctct gtgaacattc ggtttagtga acctgcaggt
     1201 cggccgccac gaccggtgcc accatggcca agcctttgtc tcaagaagaa tccaccctca
     1261 ttgaaagagc aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca
     1321 gcgcagctct ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg
     1381 ggggaccttg tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc
     1441 tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt
     1501 gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg
     1561 gacagccgac ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggcg
     1621 ctagccttgg ctccggcgag ggcaggggaa gtcttctaac atgcggggac gtggaggaaa
     1681 atcccggccc actcggcatg gactacaagg acgacgatga caagctcgat agcggcatgg
     1741 acggcggcac actgcccagg tccgcgcccc ctgcgccccc cgtccctgtc ggctgcgctg
     1801 cccggcggag acccgcgtcc ccggaactgt tgcgctgcag ccggcggcgg cgaccggcca
     1861 ccgcagagac cggaggcggc gcagcggccg tagcgcggcg caatgagcgc gagcgcaacc
     1921 gcgtgaagct ggtgaacttg ggcttccagg cgctgcggca gcacgtgccg cacggcggcg
     1981 ccagcaagaa gctgagcaag gtggagacgc tgcgctcagc cgtggagtac atccgcgcgc
     2041 tgcagcgcct gctggccgag cacgacgccg tgcgcaacgc gctggcggga gggctgaggc
     2101 cgcaggccgt gcggccgtct gcgccccgcg ggccgccagg gaccaccccg gtcgccgcct
     2161 cgccctcccg cgcttcttcg tccccgggcc gcgggggcag ctcggagccc ggctccccgc
     2221 gttccgccta ctcgtcggac gacagcggct gcgaaggcgc gctgagtcct gcggagcgcg
     2281 agctactcga cttctccagc tggttagggg gctactgagc gccctcgagc tagagttcgc
     2341 gtctggaaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     2401 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     2461 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     2521 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     2581 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     2641 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     2701 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
     2761 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     2821 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     2881 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct
     2941 ggaattaatt ctggctaagc acttcgtggc cgtcgatcgt ttaaagggag gtagtgagtc
     3001 gagatatcga tactagtgat ccagacatga taagatacat tgatgagttt ggacaaacca
     3061 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     3121 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     3181 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     3241 gtatggctga ttatgatcct ctagagggac agcccccccc caaagccccc agggatgtaa
     3301 ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc tccggtccgg
     3361 cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac ggggaaggtg
     3421 gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca gacacctggg
     3481 gggatacggg gaaaaagcca taccaatgga gctcacgggg acagcccccc cccaaagccc
     3541 ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc
     3601 gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc
     3661 acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg
     3721 cagacacctg gggggatacg gggaaaaaga attcgataaa agttttgtta ctttatagaa
     3781 gaaattttga gtttttgttt tttttaataa ataaataaac ataaataaat tgtttgttga
     3841 atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa
     3901 ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacacat gattatcttt
     3961 aacgtacgtc acaatatgat tatctttcta gggttaatct agctgcgtgt tctgcagcgt
     4021 gtcgagcatc ttcatctgct ccatcacgct gtaaaacaca tttgcaccgc gagtctgccc
     4081 gtcctccacg ggttcaaaaa cgtgaatgaa cgaggcgcgc tcactggccg tcgttttaca
     4141 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     4201 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     4261 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4321 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     4381 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4441 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4501 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4561 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4621 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4681 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     4741 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     4801 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     4861 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     4921 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     4981 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5041 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5101 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5161 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5221 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5281 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5341 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     5401 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     5461 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     5521 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     5581 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     5641 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     5701 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     5761 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5821 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5881 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5941 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6001 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6061 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6121 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6181 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6241 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6301 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6361 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6421 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6481 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6541 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     6601 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     6661 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     6721 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     6781 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     6841 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
     6901 aagcgcgccc gccgggtaac tcacggggta tccatgtcca tttctgcggc atccagccag
     6961 gatacccgtc ctcgctgacg taatatccca gcgccgcacc gctgtcatta atctgcacac
     7021 cggcacggca gttccggctg tcgccggtat tgttcgggtt gctgatgcgc ttcgggctga
     7081 ccatccggaa ctgtgtccgg aaaagccgcg acgaactggt atcccaggtg gcctgaacga
     7141 acagttcacc gttaaaggcg tgcatggcca caccttcccg aatcatcatg gtaaacgtgc
     7201 gttttcgctc aacgtcaatg cagcagcagt catcctcggc aaactctttc catgccgctt
     7261 caacctcgcg ggaaaaggca cgggcttctt cctccccgat gcccagatag cgccagcttg
     7321 ggcgatgact gagccggaaa aaagacccga cgatatgatc ctgatgcagc tagattaacc
     7381 ctagaaagat agtctgcgta aaattgacgc atg
//
