LOCUS       sequence-256354-        6767 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 6767)
  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 6767)
  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..6767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..39
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(308..324)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(308..324)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     enhancer        637..940
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        941..1144
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1095..1115
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1182..1191
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1188..1583
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1602..1655
                     /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             1665..1688
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1744..2332
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1797..1817)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2215..2226)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2412..2546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2436..2455)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2490..2509
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3324..3386
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3516..3533)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3516..3532)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3525..3547)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3674..4129
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3761..3780)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3971..3992
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4155..4259
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4260..4328,4329..5120)
                     /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(4478..4497)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5291..5879
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5780..5799
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6033..6050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6167..6188
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6203..6208,6209..6226,6227..6233)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6241..6257
                     /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     6246..6268
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6265..6281
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6265..6281
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
       61 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
      121 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
      181 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
      241 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
      301 tgttatagat ccactagttc tagagggaca gccccccccc aaagccccca gggatgtaat
      361 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
      421 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
      481 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
      541 ggatacgggg aaaaggcctc caaggcctgc aggcattagt tattaatagt aatcaattac
      601 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      661 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      721 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      781 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      841 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      901 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      961 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     1021 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     1081 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     1141 agctggttta gtgaacctgc aggtcggccg ccacgaccgg tgccaccatg gccaagcctt
     1201 tgtctcaaga agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca
     1261 tctctgaaga ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg
     1321 gtgtcaatgt atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg
     1381 ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg
     1441 gcatcttgag cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca
     1501 aagccatagt gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc
     1561 cctctggtta tgtgtgggag ggcgctagcc ttggctccgg cgagggcagg ggaagtcttc
     1621 taacatgcgg ggacgtggag gaaaatcccg gcccactcgg catggactac aaggacgacg
     1681 atgacaagct cgatagcggc atgagacgga tagctcgtct cagctagagt tcgcgtctgg
     1741 aacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     1801 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     1861 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     1921 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     1981 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     2041 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     2101 ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg
     2161 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     2221 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     2281 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggaatt
     2341 aattctggct aagcacttcg tggccgtcga tcgtttaaag ggaggtagtg agtcgagata
     2401 tcgatactag tgatccagac atgataagat acattgatga gtttggacaa accacaacta
     2461 gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa
     2521 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg
     2581 ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg
     2641 ctgattatga tcctctagag ggacagcccc cccccaaagc ccccagggat gtaattacgt
     2701 ccctcccccg ctagggggca gcagcgagcc gcccggggct ccgctccggt ccggcgctcc
     2761 ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg gcacggggaa ggtggcacgg
     2821 gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc tgcagacacc tggggggata
     2881 cggggaaaag ccataccaat ggagctcacg gggacagccc ccccccaaag cccccaggga
     2941 tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg
     3001 tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
     3061 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     3121 ctggggggat acggggaaaa agaattcgat aaaagttttg ttactttata gaagaaattt
     3181 tgagtttttg ttttttttaa taaataaata aacataaata aattgtttgt tgaatttatt
     3241 attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa taaataaacc
     3301 tcgatataca gaccgataaa acacatgcgt caattttaca catgattatc tttaacgtac
     3361 gtcacaatat gattatcttt ctagggttaa tctagctgcg tgttctgcag cgtgtcgagc
     3421 atcttcatct gctccatcac gctgtaaaac acatttgcac cgcgagtctg cccgtcctcc
     3481 acgggttcaa aaacgtgaat gaacgaggcg cgctcactgg ccgtcgtttt acaacgtcgt
     3541 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     3601 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     3661 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3721 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3781 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3841 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3901 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3961 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4021 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4081 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4141 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4201 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     4261 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     4321 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     4381 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     4441 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     4501 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     4561 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     4621 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     4681 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4741 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4801 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4861 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4921 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4981 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     5041 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     5101 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     5161 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     5221 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5281 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5341 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5401 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     5461 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5521 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5581 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5641 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5701 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5761 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5821 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5881 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5941 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     6001 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     6061 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     6121 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     6181 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     6241 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     6301 cccgccgggt aactcacggg gtatccatgt ccatttctgc ggcatccagc caggataccc
     6361 gtcctcgctg acgtaatatc ccagcgccgc accgctgtca ttaatctgca caccggcacg
     6421 gcagttccgg ctgtcgccgg tattgttcgg gttgctgatg cgcttcgggc tgaccatccg
     6481 gaactgtgtc cggaaaagcc gcgacgaact ggtatcccag gtggcctgaa cgaacagttc
     6541 accgttaaag gcgtgcatgg ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg
     6601 ctcaacgtca atgcagcagc agtcatcctc ggcaaactct ttccatgccg cttcaacctc
     6661 gcgggaaaag gcacgggctt cttcctcccc gatgcccaga tagcgccagc ttgggcgatg
     6721 actgagccgg aaaaaagacc cgacgatatg atcctgatgc agctaga
//
