LOCUS       sequence-267399-        6768 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 6768)
  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 6768)
  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-CBtW
FEATURES             Location/Qualifiers
     source          1..6768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(15..20,21..38,39..45)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    53..69
                     /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     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   584..618
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(887..903)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(887..903)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     enhancer        1216..1519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1520..1723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1674..1694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1761..1770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1767..2162
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             2181..2234
                     /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             2244..2267
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2323..2911
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2376..2396)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2794..2805)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2991..3125
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3015..3034)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3069..3088
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3904..3966
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4096..4113)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4096..4112)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4105..4127)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4254..4709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4341..4360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4551..4572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4735..4839
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4840..4908,4909..5700)
                     /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(5058..5077)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5871..6459
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6360..6379
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6613..6630
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6747..6768
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcccgccgg
      121 gtaactcacg gggtatccat gtccatttct gcggcatcca gccaggatac ccgtcctcgc
      181 tgacgtaata tcccagcgcc gcaccgctgt cattaatctg cacaccggca cggcagttcc
      241 ggctgtcgcc ggtattgttc gggttgctga tgcgcttcgg gctgaccatc cggaactgtg
      301 tccggaaaag ccgcgacgaa ctggtatccc aggtggcctg aacgaacagt tcaccgttaa
      361 aggcgtgcat ggccacacct tcccgaatca tcatggtaaa cgtgcgtttt cgctcaacgt
      421 caatgcagca gcagtcatcc tcggcaaact ctttccatgc cgcttcaacc tcgcgggaaa
      481 aggcacgggc ttcttcctcc ccgatgccca gatagcgcca gcttgggcga tgactgagcc
      541 ggaaaaaaga cccgacgata tgatcctgat gcagctagat taaccctaga aagatagtct
      601 gcgtaaaatt gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc
      661 cgtcgctgtg catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt
      721 caatgcggta agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat
      781 gattatcttt tacgtgactt ttaagattta actcatacga taattatatt gttatttcat
      841 gttctactta cgtgataact tattatatat atattttctt gttatagatc cactagttct
      901 agagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
      961 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     1021 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     1081 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc
     1141 aaggcctgca ggcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
     1201 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1261 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1321 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1381 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1441 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1501 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1561 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1621 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1681 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaacctgca
     1741 ggtcggccgc cacgaccggt gccaccatgg ccaagccttt gtctcaagaa gaatccaccc
     1801 tcattgaaag agcaacggct acaatcaaca gcatccccat ctctgaagac tacagcgtcg
     1861 ccagcgcagc tctctctagc gacggccgca tcttcactgg tgtcaatgta tatcatttta
     1921 ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc tgctgctgcg gcagctggca
     1981 acctgacttg tatcgtcgcg atcggaaatg agaacagggg catcttgagc ccctgcggac
     2041 ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa agccatagtg aaggacagtg
     2101 atggacagcc gacggcagtt gggattcgtg aattgctgcc ctctggttat gtgtgggagg
     2161 gcgctagcct tggctccggc gagggcaggg gaagtcttct aacatgcggg gacgtggagg
     2221 aaaatcccgg cccactcggc atggactaca aggacgacga tgacaagctc gatagcggca
     2281 tgagacggat agctcgtctc agctagagtt cgcgtctgga acaatcaacc tctggattac
     2341 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     2401 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     2461 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     2521 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     2581 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     2641 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     2701 gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg
     2761 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     2821 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     2881 agtcggatct ccctttgggc cgcctccccg cctggaatta attctggcta agcacttcgt
     2941 ggccgtcgat cgtttaaagg gaggtagtga gtcgagatat cgatactagt gatccagaca
     3001 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3061 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3121 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     3181 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat cctctagagg
     3241 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag
     3301 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca
     3361 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct
     3421 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag ccataccaat
     3481 ggagctcacg gggacagccc ccccccaaag cccccaggga tgtaattacg tccctccccc
     3541 gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc
     3601 cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg ggatcgcttt
     3661 cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa
     3721 agaattcgat aaaagttttg ttactttata gaagaaattt tgagtttttg ttttttttaa
     3781 taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat
     3841 ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa
     3901 acacatgcgt caattttaca catgattatc tttaacgtac gtcacaatat gattatcttt
     3961 ctagggttaa tctagctgcg tgttctgcag cgtgtcgagc atcttcatct gctccatcac
     4021 gctgtaaaac acatttgcac cgcgagtctg cccgtcctcc acgggttcaa aaacgtgaat
     4081 gaacgaggcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     4141 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     4201 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     4261 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4321 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4381 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4441 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4501 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     4561 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     4621 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     4681 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     4741 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     4801 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     4861 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     4921 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     4981 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     5041 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     5101 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     5161 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     5221 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     5281 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     5341 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     5401 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     5461 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     5521 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     5581 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     5641 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     5701 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     5761 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     5821 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5881 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5941 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6001 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     6061 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6121 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6181 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6241 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6301 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6361 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6421 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6481 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     6541 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     6601 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     6661 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     6721 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcat
//
