LOCUS       sequence-256374-        8352 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 8352)
  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 8352)
  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-ChiNW-ASCL2
FEATURES             Location/Qualifiers
     source          1..8352
                     /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"
     regulatory      1273..1282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    join(1911..2485,2486..2488,2489..2497)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2078..2095)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2305..2324
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2510..3304
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLAATGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2564..2583)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3174..3193
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     misc_feature    3329..3917
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3382..3402)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3800..3811)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3997..4131
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4021..4040)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4075..4094
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4909..4971
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5101..5118)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5101..5117)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5110..5132)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5259..5714
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5346..5365)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5556..5577
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5740..5844
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5845..5913,5914..6705)
                     /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(6063..6082)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6876..7464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7365..7384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7618..7635
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7752..7773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7788..7793,7794..7811,7812..7818)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7826..7842
                     /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     7831..7853
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7850..7866
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7850..7866
                     /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 aggagtcaat gggaaaaacc cattggagcc
      601 aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag
      661 ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc
      721 caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt
      781 actgacatgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt
      841 caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga
      901 ctttccattg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc
      961 ccattattgg cacatacata aggtcaatag gggtgactag tggagaagag catgcttgag
     1021 ggctgagtgc ccctcagtgg gcagagagca catggcccac agtccctgag aagttggggg
     1081 gaggggtggg caattgaact ggtgcctaga gaaggtgggg cttgggtaaa ctgggaaagt
     1141 gatgtggtgt actggctcca cctttttccc cagggtgggg gagaaccata tataagtgca
     1201 gtagtctctg tgaacattcg gtttagtgaa cctgcaggtc ggccgccacg accggtgcct
     1261 actctagagc tagccaccat ggacggcggc acactgccca ggtccgcgcc ccctgcgccc
     1321 cccgtccctg tcggctgcgc tgcccggcgg agacccgcgt ccccggaact gttgcgctgc
     1381 agccggcggc ggcgaccggc caccgcagag accggaggcg gcgcagcggc cgtagcgcgg
     1441 cgcaatgagc gcgagcgcaa ccgcgtgaag ctggtgaact tgggcttcca ggcgctgcgg
     1501 cagcacgtgc cgcacggcgg cgccagcaag aagctgagca aggtggagac gctgcgctca
     1561 gccgtggagt acatccgcgc gctgcagcgc ctgctggccg agcacgacgc cgtgcgcaac
     1621 gcgctggcgg gagggctgag gccgcaggcc gtgcggccgt ctgcgccccg cgggccgcca
     1681 gggaccaccc cggtcgccgc ctcgccctcc cgcgcttctt cgtccccggg ccgcgggggc
     1741 agctcggagc ccggctcccc gcgttccgcc tactcgtcgg acgacagcgg ctgcgaaggc
     1801 gcgctgagtc ctgcggagcg cgagctactc gacttctcca gctggttagg gggctactga
     1861 gcgccctcga cctatgagcc tatcgaattt aaatcggatc cgcggccgcg cccctctccc
     1921 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc
     1981 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc
     2041 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt
     2101 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct
     2161 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa
     2221 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt
     2281 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag
     2341 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt
     2401 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt
     2461 ttcctttgaa aaacacgatg ataatatggc cacaaccatg gcgtccggaa tgattgaaca
     2521 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     2581 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     2641 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     2701 agcgcggcta tcgtggctgg ccgcgacggg cgttccttgc gcagctgtgc tcgacgttgt
     2761 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     2821 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     2881 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     2941 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3001 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     3061 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     3121 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     3181 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     3241 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     3301 ctgagtcgct agagttcgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag
     3361 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3421 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3481 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3541 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3601 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3661 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3721 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3781 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3841 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3901 ttgggccgcc tccccgcctg gaattaattc tggctaagca cttcgtggcc gtcgatcgtt
     3961 taaagggagg tagtgagtcg agatatcgat actagtgatc cagacatgat aagatacatt
     4021 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     4081 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     4141 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     4201 taaaacctct acaaatgtgg tatggctgat tatgatcctc tagagggaca gccccccccc
     4261 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     4321 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     4381 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     4441 gagcctgcag acacctgggg ggatacgggg aaaagccata ccaatggagc tcacggggac
     4501 agcccccccc caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag
     4561 cgagccgccc ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg
     4621 tgcggggaca gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct
     4681 cgctgctctt tgagcctgca gacacctggg gggatacggg gaaaaagaat tcgataaaag
     4741 ttttgttact ttatagaaga aattttgagt ttttgttttt tttaataaat aaataaacat
     4801 aaataaattg tttgttgaat ttattattag tatgtaagtg taaatataat aaaacttaat
     4861 atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca tgcgtcaatt
     4921 ttacacatga ttatctttaa cgtacgtcac aatatgatta tctttctagg gttaatctag
     4981 ctgcgtgttc tgcagcgtgt cgagcatctt catctgctcc atcacgctgt aaaacacatt
     5041 tgcaccgcga gtctgcccgt cctccacggg ttcaaaaacg tgaatgaacg aggcgcgctc
     5101 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     5161 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     5221 cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt
     5281 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     5341 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5401 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5461 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5521 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5581 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     5641 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5701 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5761 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5821 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5881 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5941 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6001 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6061 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6121 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6181 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6241 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6301 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6361 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6421 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6481 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6541 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6601 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6661 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6721 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6781 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6841 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6901 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6961 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7021 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7081 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7141 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7201 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7261 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7321 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7381 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7441 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7501 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7561 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7621 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7681 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7741 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7801 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     7861 tatgaccatg attacgccaa gcgcgcccgc cgggtaactc acggggtatc catgtccatt
     7921 tctgcggcat ccagccagga tacccgtcct cgctgacgta atatcccagc gccgcaccgc
     7981 tgtcattaat ctgcacaccg gcacggcagt tccggctgtc gccggtattg ttcgggttgc
     8041 tgatgcgctt cgggctgacc atccggaact gtgtccggaa aagccgcgac gaactggtat
     8101 cccaggtggc ctgaacgaac agttcaccgt taaaggcgtg catggccaca ccttcccgaa
     8161 tcatcatggt aaacgtgcgt tttcgctcaa cgtcaatgca gcagcagtca tcctcggcaa
     8221 actctttcca tgccgcttca acctcgcggg aaaaggcacg ggcttcttcc tccccgatgc
     8281 ccagatagcg ccagcttggg cgatgactga gccggaaaaa agacccgacg atatgatcct
     8341 gatgcagcta ga
//
