LOCUS       sequence-268573-        8384 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 8384)
  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 8384)
  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-ChPtinGW
FEATURES             Location/Qualifiers
     source          1..8384
                     /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"
     CDS             1836..1889
                     /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             1896..1922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1896..1922
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    1965..2531
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1981..2533
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2125..2142)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2352..2371
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2545..2547,2548..2550,2551..3261)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2590..2611)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2851..2870)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3198..3219
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3265..3285
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3321..3909
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3374..3394)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3792..3803)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3989..4123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4013..4032)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4067..4086
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4902..4964
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5094..5111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5094..5110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5103..5125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5252..5707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5339..5358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5549..5570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5733..5837
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5838..5906,5907..6698)
                     /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(6056..6075)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6869..7457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7358..7377
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7611..7628
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7745..7766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7781..7786,7787..7804,7805..7811)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7819..7835
                     /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     7824..7846
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7843..7859
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7843..7859
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   8350..8384
                     /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 accatgactg aatacaaacc aactgttcgc ctggcaactc
     1261 gtgatgatgt tccacgtgca gttcgcaccc tggctgctgc atttgctgac taccctgcaa
     1321 cccgtcacac tgtggaccca gaccgccaca ttgaacgtgt gactgaactg caggagctgt
     1381 tcctgacccg tgtgggcctg gacattggca aagtgtgggt ggcagatgat ggtgctgctg
     1441 tggcagtgtg gaccacccct gaatctgttg aagctggtgc agtgtttgct gagattggcc
     1501 cacgcatggc agaactgtct ggcagccgcc tggcagcaca acagcagatg gaaggtctgc
     1561 tggcaccaca ccgcccaaaa gaacctgctt ggttcctggc aactgtgggt gtgagccctg
     1621 accaccaggg taagggcctg ggctctgcag tggtgctgcc tggtgtggaa gcagctgaac
     1681 gtgcaggtgt gcctgctttc ctggagacct cagctccacg caacctgcct ttctatgaac
     1741 gcctgggctt cactgtgact gctgatgtgg aagtgccaga aggcccacgc acttggtgca
     1801 tgactcgcaa accaggtgct agccttggct ccggcgaggg caggggaagt cttctaacat
     1861 gcggggacgt ggaggaaaat cccggcccac tcggctaccc ctacgacgtg cccgactacg
     1921 ccagcggcat gagacggata gctcgtctca gctagaatag gaattccgcc ccccccccta
     1981 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     2041 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     2101 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     2161 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     2221 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     2281 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     2341 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     2401 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     2461 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     2521 cacgatgata atatggccac aaccatggtg agcaagggcg aggagctgtt caccggggtg
     2581 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc
     2641 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2701 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2761 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2821 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2881 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2941 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3001 atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
     3061 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3121 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3181 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3241 ggcatggacg agctgtacaa gggccccaag aagaagcgca aggtgggcgc ctaagtacag
     3301 ctagagttcg cgtctggaac aatcaacctc tggattacaa aatttgtgaa agattgactg
     3361 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     3421 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     3481 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     3541 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     3601 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     3661 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaagctga
     3721 cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     3781 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     3841 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     3901 cctccccgcc tggaattaat tctggctaag cacttcgtgg ccgtcgatcg tttaaaggga
     3961 ggtagtgagt cgagatatcg atactagtga tccagacatg ataagataca ttgatgagtt
     4021 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     4081 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     4141 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     4201 ctacaaatgt ggtatggctg attatgatcc tctagaggga cagccccccc ccaaagcccc
     4261 cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc cggggctccg
     4321 ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac agcccgggca
     4381 cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct ttgagcctgc
     4441 agacacctgg ggggatacgg ggaaaaagcc ataccaatgg agctcacggg gacagccccc
     4501 ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag cagcgagccg
     4561 cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca gcgtgcgggg
     4621 acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct tctcgctgct
     4681 ctttgagcct gcagacacct ggggggatac ggggaaaaag aattcgataa aagttttgtt
     4741 actttataga agaaattttg agtttttgtt ttttttaata aataaataaa cataaataaa
     4801 ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt aatatctatt
     4861 caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca attttacaca
     4921 tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc tagctgcgtg
     4981 ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc tgtaaaacac atttgcaccg
     5041 cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga acgaggcgcg ctcactggcc
     5101 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     5161 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     5221 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     5281 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5341 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5401 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5461 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5521 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5581 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5641 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5701 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5761 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5821 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5881 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5941 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6001 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6061 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6121 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6181 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6241 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6301 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6361 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6421 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6481 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6541 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6601 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6661 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6721 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6781 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6841 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6901 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6961 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7021 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7081 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7141 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7201 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7261 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7321 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7381 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7441 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7501 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7561 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7621 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7681 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7741 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     7801 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     7861 atgattacgc caagcgcgcc cgccgggtaa ctcacggggt atccatgtcc atttctgcgg
     7921 catccagcca ggatacccgt cctcgctgac gtaatatccc agcgccgcac cgctgtcatt
     7981 aatctgcaca ccggcacggc agttccggct gtcgccggta ttgttcgggt tgctgatgcg
     8041 cttcgggctg accatccgga actgtgtccg gaaaagccgc gacgaactgg tatcccaggt
     8101 ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc acaccttccc gaatcatcat
     8161 ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag tcatcctcgg caaactcttt
     8221 ccatgccgct tcaacctcgc gggaaaaggc acgggcttct tcctccccga tgcccagata
     8281 gcgccagctt gggcgatgac tgagccggaa aaaagacccg acgatatgat cctgatgcag
     8341 ctagattaac cctagaaaga tagtctgcgt aaaattgacg catg
//
