LOCUS       sequence-268594-        8342 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 8342)
  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 8342)
  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-PLAGL2
FEATURES             Location/Qualifiers
     source          1..8342
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      374..383
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             380..775
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             794..847
                     /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             857..880
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2435..3023
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2488..2508)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2906..2917)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3103..3237
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3127..3146)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3181..3200
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4016..4078
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4208..4225)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4208..4224)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4217..4239)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4366..4821
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4453..4472)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4663..4684
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4847..4951
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4952..5020,5021..5812)
                     /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(5170..5189)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5983..6571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6472..6491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6725..6742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6859..6880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6895..6900,6901..6918,6919..6925)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6933..6949
                     /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     6938..6960
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6957..6973
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6957..6973
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   7464..7498
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7767..7783)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7767..7783)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
ORIGIN
        1 cactgagtca atagggactt tccattgggt tttgcccagt acaaaaggtc aatagggggt
       61 gagtcaatgg gtttttccca ttattggcac atacataagg tcaatagggg tgactagtgg
      121 agaagagcat gcttgagggc tgagtgcccc tcagtgggca gagagcacat ggcccacagt
      181 ccctgagaag ttggggggag gggtgggcaa ttgaactggt gcctagagaa ggtggggctt
      241 gggtaaactg ggaaagtgat gtggtgtact ggctccacct ttttccccag ggtgggggag
      301 aaccatatat aagtgcagta gtctctgtga acattcggtt tagtgaacct gcaggtcggc
      361 cgccacgacc ggtgccacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga
      421 aagagcaacg gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc
      481 agctctctct agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg
      541 accttgtgca gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac
      601 ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg
      661 acaggtgctt ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca
      721 gccgacggca gttgggattc gtgaattgct gccctctggt tatgtgtggg agggcgctag
      781 ccttggctcc ggcgagggca ggggaagtct tctaacatgc ggggacgtgg aggaaaatcc
      841 cggcccactc ggcatggact acaaggacga cgatgacaag ctcgatagcg gcatgaccac
      901 atttttcacc agcgtccccc cctggattca agatgcaaag caggaggagg aagtgggctg
      961 gaaactagtt cccaggcctc ggggccggga ggcggagagt caagtgaagt gccaatgtga
     1021 aatttcggga acacctttct caaatgggga gaagctgagg cctcacagcc tcccgcaacc
     1081 agagcagaga ccatatagct gccctcagct gcactgtggc aaggcttttg cttccaaata
     1141 caagctgtat aggcacatgg ccacccactc agcccagaaa ccccaccagt gtatgtactg
     1201 tgataagatg tttcaccgca aggaccatct gcggaaccat ctgcagaccc atgatcctaa
     1261 caaagaggcc ctccactgct ctgagtgcgg taagaattac aatacgaagc tgggctaccg
     1321 gcgccacctg gccatgcatg ctgccagcag cggtgacctc agctgcaagg tgtgcctgca
     1381 gacctttgag agtacccagg ccctgctaga gcacctgaag gcccactcac gccgggtagc
     1441 aggcggtgcc aaggagaaga agcacccctg tgaccactgc gaccggcggt tctatactcg
     1501 taaggatgta cggcggcacc tagtggtgca cacaggccgt aaggacttcc tgtgccagta
     1561 ctgtgcccag cggtttggcc gtaaggacca cctgacgcgt catgtcaaga agagccactc
     1621 gcaggagctg ctcaagatca agacagagcc cgtggacatg ttaggcctac tcagctgcag
     1681 ctccacagtc agtgtgaagg aagagctgag ccctgtgctg tgcatggcct ctcgggacgt
     1741 aatggggacc aaggccttcc ctggcatgtt gcccatgggc atgtatggtg cccacatccc
     1801 taccatgccc agcacgggcg tgccacactc cctggtgcac aacacgctgc ccatgggtat
     1861 gagctaccct ctggaatcct cacctatctc ttccccagct cagctccctc caaaatacca
     1921 gcttggatct acctcatact tgcccgacaa attgcccaaa gtggaggtgg atagttttct
     1981 ggcggagctt cctggaagcc tgtctctctc atccgctgaa ccccagcccg cctcacctca
     2041 gccggcggca gctgcggccc tcctagatga agcactgctt gccaagagcc ccgccaacct
     2101 ctctgaggcc ctctgcgctg ctaatgtgga cttctcccac ctactgggct ttcttccact
     2161 caacctgccc ccgtgtaacc cacctggggc cacaggaggc ctggtcatgg gctactccca
     2221 ggctgaggca cagcccctgc ttaccacttt gcaagctcag cctcaagatt ccccaggagc
     2281 tgggggacca ctgaactttg ggcctctgca ctccttgcct cctgtcttca cgtctggcct
     2341 gagtagcacc accctgcctc gtttccatca agcattccag tagcccccac agaggccctc
     2401 agctcagttt ttggctagag ttcgcgtctg gaacaatcaa cctctggatt acaaaatttg
     2461 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     2521 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     2581 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     2641 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     2701 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     2761 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     2821 gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg
     2881 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     2941 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3001 ctccctttgg gccgcctccc cgcctggaat taattctggc taagcacttc gtggccgtcg
     3061 atcgtttaaa gggaggtagt gagtcgagat atcgatacta gtgatccaga catgataaga
     3121 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3181 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3241 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     3301 agcaagtaaa acctctacaa atgtggtatg gctgattatg atcctctaga gggacagccc
     3361 ccccccaaag cccccaggga tgtaattacg tccctccccc gctagggggc agcagcgagc
     3421 cgcccggggc tccgctccgg tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg
     3481 ggacagcccg ggcacgggga aggtggcacg ggatcgcttt cctctgaacg cttctcgctg
     3541 ctctttgagc ctgcagacac ctggggggat acggggaaaa agccatacca atggagctca
     3601 cggggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg
     3661 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc
     3721 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa
     3781 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaagaattcg
     3841 ataaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt aataaataaa
     3901 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     3961 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     4021 gtcaatttta cacatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     4081 aatctagctg cgtgttctgc agcgtgtcga gcatcttcat ctgctccatc acgctgtaaa
     4141 acacatttgc accgcgagtc tgcccgtcct ccacgggttc aaaaacgtga atgaacgagg
     4201 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4261 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4321 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     4381 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4441 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4501 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4561 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4621 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4681 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4741 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4801 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4861 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4921 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     4981 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5041 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5101 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5161 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5221 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5281 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5341 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5401 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5461 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5521 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5581 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5641 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5701 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5761 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5821 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5881 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5941 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6001 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6061 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6121 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6181 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6241 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6301 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6361 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6421 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6481 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6541 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6601 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6661 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6721 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     6781 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     6841 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     6901 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     6961 aaacagctat gaccatgatt acgccaagcg cgcccgccgg gtaactcacg gggtatccat
     7021 gtccatttct gcggcatcca gccaggatac ccgtcctcgc tgacgtaata tcccagcgcc
     7081 gcaccgctgt cattaatctg cacaccggca cggcagttcc ggctgtcgcc ggtattgttc
     7141 gggttgctga tgcgcttcgg gctgaccatc cggaactgtg tccggaaaag ccgcgacgaa
     7201 ctggtatccc aggtggcctg aacgaacagt tcaccgttaa aggcgtgcat ggccacacct
     7261 tcccgaatca tcatggtaaa cgtgcgtttt cgctcaacgt caatgcagca gcagtcatcc
     7321 tcggcaaact ctttccatgc cgcttcaacc tcgcgggaaa aggcacgggc ttcttcctcc
     7381 ccgatgccca gatagcgcca gcttgggcga tgactgagcc ggaaaaaaga cccgacgata
     7441 tgatcctgat gcagctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca
     7501 ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac
     7561 atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg
     7621 attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt
     7681 ttaagattta actcatacga taattatatt gttatttcat gttctactta cgtgataact
     7741 tattatatat atattttctt gttatagatc cactagttct agagggacag ccccccccca
     7801 aagcccccag ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg
     7861 ggctccgctc cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc
     7921 ccgggcacgg ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg
     7981 agcctgcaga cacctggggg gatacgggga aaaggcctcc aaggcctgca ggagtcaatg
     8041 ggaaaaaccc attggagcca agtacactga ctcaataggg actttccatt gggttttgcc
     8101 cagtacataa ggtcaatagg gggtgagtca acaggaaagt cccattggag ccaagtacat
     8161 tgagtcaata gggactttcc aatgggtttt gcccagtaca taaggtcaat gggaggtaag
     8221 ccaatgggtt tttcccatta ctgacatgta tactgagtca ttagggactt tccaatgggt
     8281 tttgcccagt acataaggtc aataggggtg aatcaacagg aaagtcccat tggagccaag
     8341 ta
//
