LOCUS       sequence-267551-        7373 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 7373)
  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 7373)
  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-IGF2
FEATURES             Location/Qualifiers
     source          1..7373
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     8..24
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8..24
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   515..549
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(818..834)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(818..834)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     regulatory      1767..1776
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1773..2168
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             2187..2240
                     /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             2250..2273
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2859..3447
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2912..2932)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3330..3341)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3527..3661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3551..3570)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3605..3624
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4440..4502
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4632..4649)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4632..4648)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4641..4663)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4790..5245
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4877..4896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5087..5108
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5271..5375
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5376..5444,5445..6236)
                     /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(5594..5613)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6407..6995
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6896..6915
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7149..7166
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7283..7304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7319..7324,7325..7342,7343..7349)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7357..7373
                     /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     join(7362..7373,1..11)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcccgccg ggtaactcac
       61 ggggtatcca tgtccatttc tgcggcatcc agccaggata cccgtcctcg ctgacgtaat
      121 atcccagcgc cgcaccgctg tcattaatct gcacaccggc acggcagttc cggctgtcgc
      181 cggtattgtt cgggttgctg atgcgcttcg ggctgaccat ccggaactgt gtccggaaaa
      241 gccgcgacga actggtatcc caggtggcct gaacgaacag ttcaccgtta aaggcgtgca
      301 tggccacacc ttcccgaatc atcatggtaa acgtgcgttt tcgctcaacg tcaatgcagc
      361 agcagtcatc ctcggcaaac tctttccatg ccgcttcaac ctcgcgggaa aaggcacggg
      421 cttcttcctc cccgatgccc agatagcgcc agcttgggcg atgactgagc cggaaaaaag
      481 acccgacgat atgatcctga tgcagctaga ttaaccctag aaagatagtc tgcgtaaaat
      541 tgacgcatgc attcttgaaa tattgctctc tctttctaaa tagcgcgaat ccgtcgctgt
      601 gcatttagga catctcagtc gccgcttgga gctcccgtga ggcgtgcttg tcaatgcggt
      661 aagtgtcact gattttgaac tataacgacc gcgtgagtca aaatgacgca tgattatctt
      721 ttacgtgact tttaagattt aactcatacg ataattatat tgttatttca tgttctactt
      781 acgtgataac ttattatata tatattttct tgttatagat ccactagttc tagagggaca
      841 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
      901 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
      961 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     1021 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaggcctc caaggcctgc
     1081 aggagtcaat gggaaaaacc cattggagcc aagtacactg actcaatagg gactttccat
     1141 tgggttttgc ccagtacata aggtcaatag ggggtgagtc aacaggaaag tcccattgga
     1201 gccaagtaca ttgagtcaat agggactttc caatgggttt tgcccagtac ataaggtcaa
     1261 tgggaggtaa gccaatgggt ttttcccatt actgacatgt atactgagtc attagggact
     1321 ttccaatggg ttttgcccag tacataaggt caataggggt gaatcaacag gaaagtccca
     1381 ttggagccaa gtacactgag tcaataggga ctttccattg ggttttgccc agtacaaaag
     1441 gtcaataggg ggtgagtcaa tgggtttttc ccattattgg cacatacata aggtcaatag
     1501 gggtgactag tggagaagag catgcttgag ggctgagtgc ccctcagtgg gcagagagca
     1561 catggcccac agtccctgag aagttggggg gaggggtggg caattgaact ggtgcctaga
     1621 gaaggtgggg cttgggtaaa ctgggaaagt gatgtggtgt actggctcca cctttttccc
     1681 cagggtgggg gagaaccata tataagtgca gtagtctctg tgaacattcg gtttagtgaa
     1741 cctgcaggtc ggccgccacg accggtgcca ccatggccaa gcctttgtct caagaagaat
     1801 ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca
     1861 gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc
     1921 attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag
     1981 ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct
     2041 gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg
     2101 acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt
     2161 gggagggcgc tagccttggc tccggcgagg gcaggggaag tcttctaaca tgcggggacg
     2221 tggaggaaaa tcccggccca ctcggcatgg actacaagga cgacgatgac aagctcgata
     2281 gcggcatggg aatcccaatg gggaagtcga tgctggtgct tctcaccttc ttggccttcg
     2341 cctcgtgctg cattgctgct taccgcccca gtgagaccct gtgcggcggg gagctggtgg
     2401 acaccctcca gttcgtctgt ggggaccgcg gcttctactt cagcaggccc gcaagccgtg
     2461 tgagccgtcg cagccgtggc atcgttgagg agtgctgttt ccgcagctgt gacctggccc
     2521 tcctggagac gtactgtgct acccccgcca agtccgagag ggacgtgtcg acccctccga
     2581 ccgtgcttcc ggacaacttc cccagatacc ccgtgggcaa gttcttccaa tatgacacct
     2641 ggaagcagtc cacccagcgc ctgcgcaggg gcctgcctgc cctcctgcgt gcccgccggg
     2701 gtcacgtgct cgccaaggag ctcgaggcgt tcagggaggc caaacgtcac cgtcccctga
     2761 ttgctctacc cacccaagac cccgcccacg ggggcgcccc cccagagatg gccagcaatc
     2821 ggaagtgagc aaaactggct agagttcgcg tctggaacaa tcaacctctg gattacaaaa
     2881 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     2941 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3001 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3061 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3121 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3181 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3241 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     3301 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3361 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3421 ggatctccct ttgggccgcc tccccgcctg gaattaattc tggctaagca cttcgtggcc
     3481 gtcgatcgtt taaagggagg tagtgagtcg agatatcgat actagtgatc cagacatgat
     3541 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     3601 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     3661 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     3721 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc tagagggaca
     3781 gccccccccc aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc
     3841 gagccgcccg gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt
     3901 gcggggacag cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc
     3961 gctgctcttt gagcctgcag acacctgggg ggatacgggg aaaaagccat accaatggag
     4021 ctcacgggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
     4081 gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg
     4141 agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc
     4201 tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaagaa
     4261 ttcgataaaa gttttgttac tttatagaag aaattttgag tttttgtttt ttttaataaa
     4321 taaataaaca taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa
     4381 taaaacttaa tatctattca aattaataaa taaacctcga tatacagacc gataaaacac
     4441 atgcgtcaat tttacacatg attatcttta acgtacgtca caatatgatt atctttctag
     4501 ggttaatcta gctgcgtgtt ctgcagcgtg tcgagcatct tcatctgctc catcacgctg
     4561 taaaacacat ttgcaccgcg agtctgcccg tcctccacgg gttcaaaaac gtgaatgaac
     4621 gaggcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     4681 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     4741 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     4801 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4861 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4921 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4981 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     5041 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     5101 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     5161 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     5221 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5281 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5341 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5401 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5461 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5521 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5581 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5641 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5701 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5761 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5821 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5881 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5941 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     6001 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6061 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6121 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6181 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6241 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6301 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6361 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6421 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6481 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6541 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6601 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6661 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6721 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6781 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6841 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6901 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6961 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     7021 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     7081 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     7141 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     7201 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7261 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7321 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caa
//
