LOCUS       sequence-267686-        6843 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 6843)
  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 6843)
  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..6843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..35
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(526..542)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(526..542)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(539..561)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    550..566
                     /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(574..580,581..598,599..604))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    619..640
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(757..774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(928..1516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1008..1027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1687..2478,2479..2547))
                     /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     2310..2329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2548..2652)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2678..3133)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2815..2836)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3027..3046
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3260..3282
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3274..3291
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3275..3291
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   3421..3483
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4262..4396
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4299..4318)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4353..4372
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    4476..5064
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             4582..4593
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     4991..5011
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5120..5143)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(5153..5206)
                     /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             complement(5225..5620)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5617..5626
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     6559..6575
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6559..6575
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctgcat caggatcata
       61 tcgtcgggtc ttttttccgg ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg
      121 aggaagaagc ccgtgccttt tcccgcgagg ttgaagcggc atggaaagag tttgccgagg
      181 atgactgctg ctgcattgac gttgagcgaa aacgcacgtt taccatgatg attcgggaag
      241 gtgtggccat gcacgccttt aacggtgaac tgttcgttca ggccacctgg gataccagtt
      301 cgtcgcggct tttccggaca cagttccgga tggtcagccc gaagcgcatc agcaacccga
      361 acaataccgg cgacagccgg aactgccgtg ccggtgtgca gattaatgac agcggtgcgg
      421 cgctgggata ttacgtcagc gaggacgggt atcctggctg gatgccgcag aaatggacat
      481 ggataccccg tgagttaccc ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc
      541 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
      601 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
      661 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
      721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
      781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     1381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1621 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1681 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     1861 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     2101 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     2221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     2281 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     2341 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     2401 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     2461 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     2521 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     2581 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2641 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
     2701 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2761 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2821 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2881 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2941 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3001 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3061 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3121 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     3181 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     3241 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcc
     3301 tcgttcattc acgtttttga acccgtggag gacgggcaga ctcgcggtgc aaatgtgttt
     3361 tacagcgtga tggagcagat gaagatgctc gacacgctgc agaacacgca gctagattaa
     3421 ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgtgta aaattgacgc
     3481 atgtgtttta tcggtctgta tatcgaggtt tatttattaa tttgaataga tattaagttt
     3541 tattatattt acacttacat actaataata aattcaacaa acaatttatt tatgtttatt
     3601 tatttattaa aaaaaacaaa aactcaaaat ttcttctata aagtaacaaa acttttatcg
     3661 aattcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt
     3721 cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg
     3781 ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc
     3841 cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccccgtg
     3901 agctccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc tcaaagagca
     3961 gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc
     4021 gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc cccgggcggc
     4081 tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct ttgggggggg
     4141 gctgtccctc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4201 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4261 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4321 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4381 ttatcatgtc tggatcacta gtatcgatat ctcgactcac tacctccctt taaacgatcg
     4441 acggccacga agtgcttagc cagaattaat tccaggcggg gaggcggccc aaagggagat
     4501 ccgactcgtc tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc
     4561 gcgggaagga aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg
     4621 cagaatccag gtggcaacac aggcgagcag ccatggaaag gacgtcagct tccccgacaa
     4681 caccacggaa ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc
     4741 ggcgatgagt tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg
     4801 acaggtggtg gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac
     4861 gccacgttgc ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata
     4921 caaggaggag aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc
     4981 agcgtatcca catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca
     5041 aattttgtaa tccagaggtt gattgttcca gacgcgaact ctagctgaga cgagctatcc
     5101 gtctcatgcc gctatcgagc ttgtcatcgt cgtccttgta gtccatgccg agtgggccgg
     5161 gattttcctc cacgtccccg catgttagaa gacttcccct gccctcgccg gagccaaggc
     5221 tagcgccctc ccacacataa ccagagggca gcaattcacg aatcccaact gccgtcggct
     5281 gtccatcact gtccttcact atggctttga tcccaggatg cagatcgaga agcacctgtc
     5341 ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt tccgatcgcg acgatacaag
     5401 tcaggttgcc agctgccgca gcagcagcag tgcccagcac cacgagttct gcacaaggtc
     5461 ccccagtaaa atgatataca ttgacaccag tgaagatgcg gccgtcgcta gagagagctg
     5521 cgctggcgac gctgtagtct tcagagatgg ggatgctgtt gattgtagcc gttgctcttt
     5581 caatgagggt ggattcttct tgagacaaag gcttggccat ggtggcaccg gtcgtggcgg
     5641 ccgacctgca ggttcactaa accgaatgtt cacagagact actgcactta tatatggttc
     5701 tcccccaccc tggggaaaaa ggtggagcca gtacaccaca tcactttccc agtttaccca
     5761 agccccacct tctctaggca ccagttcaat tgcccacccc tccccccaac ttctcaggga
     5821 ctgtgggcca tgtgctctct gcccactgag gggcactcag ccctcaagca tgctcttctc
     5881 cactagtcac ccctattgac cttatgtatg tgccaataat gggaaaaacc cattgactca
     5941 ccccctattg accttttgta ctgggcaaaa cccaatggaa agtccctatt gactcagtgt
     6001 acttggctcc aatgggactt tcctgttgat tcacccctat tgaccttatg tactgggcaa
     6061 aacccattgg aaagtcccta atgactcagt atacatgtca gtaatgggaa aaacccattg
     6121 gcttacctcc cattgacctt atgtactggg caaaacccat tggaaagtcc ctattgactc
     6181 aatgtacttg gctccaatgg gactttcctg ttgactcacc ccctattgac cttatgtact
     6241 gggcaaaacc caatggaaag tccctattga gtcagtgtac ttggctccaa tgggtttttc
     6301 ccattgactc ctgcaggcct tggaggcctt ttccccgtat ccccccaggt gtctgcaggc
     6361 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     6421 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     6481 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     6541 ttgggggggg gctgtccctc tagaactagt ggatctataa caagaaaata tatatataat
     6601 aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta
     6661 aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa
     6721 tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga
     6781 tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga
     6841 atg
//
