LOCUS       sequence-256343-        8272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for shRNA-mediated knockdown, co-expressed with 
            GFP-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8272)
  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 8272)
  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-Sh-PIR702W-Luc
FEATURES             Location/Qualifiers
     source          1..8272
                     /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"
     promoter        580..828
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     580..600
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     751..770
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2203..2256
                     /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             2263..2289
                     /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             2263..2289
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3249..3837
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3302..3322)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3720..3731)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3917..4051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3941..3960)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3995..4014
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4829..4891
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5021..5038)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5021..5037)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5030..5052)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5179..5634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5266..5285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5476..5497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5660..5764
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5765..5833,5834..6625)
                     /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(5983..6002)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6796..7384
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7285..7304
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7538..7555
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7672..7693
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7708..7713,7714..7731,7732..7738)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7746..7762
                     /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     7751..7773
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7770..7786
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7770..7786
                     /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 cagggccgcc cccttcaccg agggcctatt tcccatgatt
      601 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
      661 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      721 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      781 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg tcacagaatc
      841 gtcgtatgca gctcgagctg catacgacga ttctgtgatt tttgcctcca aggcctgcag
      901 gagtcaatgg gaaaaaccca ttggagccaa gtacactgac tcaataggga ctttccattg
      961 ggttttgccc agtacataag gtcaataggg ggtgagtcaa caggaaagtc ccattggagc
     1021 caagtacatt gagtcaatag ggactttcca atgggttttg cccagtacat aaggtcaatg
     1081 ggaggtaagc caatgggttt ttcccattac tgacatgtat actgagtcat tagggacttt
     1141 ccaatgggtt ttgcccagta cataaggtca ataggggtga atcaacagga aagtcccatt
     1201 ggagccaagt acactgagtc aatagggact ttccattggg ttttgcccag tacaaaaggt
     1261 caataggggg tgagtcaatg ggtttttccc attattggca catacataag gtcaataggg
     1321 gtgactagtg gagaagagca tgcttgaggg ctgagtgccc ctcagtgggc agagagcaca
     1381 tggcccacag tccctgagaa gttgggggga ggggtgggca attgaactgg tgcctagaga
     1441 aggtggggct tgggtaaact gggaaagtga tgtggtgtac tggctccacc tttttcccca
     1501 gggtggggga gaaccatata taagtgcagt agtctctgtg aacattcggt ttagtgaacc
     1561 tgcaggtcgg ccgccacgac cggtgccacc atgactgaat acaaaccaac tgttcgcctg
     1621 gcaactcgtg atgatgttcc acgtgcagtt cgcaccctgg ctgctgcatt tgctgactac
     1681 cctgcaaccc gtcacactgt ggacccagac cgccacattg aacgtgtgac tgaactgcag
     1741 gagctgttcc tgacccgtgt gggcctggac attggcaaag tgtgggtggc agatgatggt
     1801 gctgctgtgg cagtgtggac cacccctgaa tctgttgaag ctggtgcagt gtttgctgag
     1861 attggcccac gcatggcaga actgtctggc agccgcctgg cagcacaaca gcagatggaa
     1921 ggtctgctgg caccacaccg cccaaaagaa cctgcttggt tcctggcaac tgtgggtgtg
     1981 agccctgacc accagggtaa gggcctgggc tctgcagtgg tgctgcctgg tgtggaagca
     2041 gctgaacgtg caggtgtgcc tgctttcctg gagacctcag ctccacgcaa cctgcctttc
     2101 tatgaacgcc tgggcttcac tgtgactgct gatgtggaag tgccagaagg cccacgcact
     2161 tggtgcatga ctcgcaaacc aggtgctagc cttggctccg gcgagggcag gggaagtctt
     2221 ctaacatgcg gggacgtgga ggaaaatccc ggcccactcg gctaccccta cgacgtgccc
     2281 gactacgcca gcggcatggc cagaaaggtg gatctgacaa gctgtgacag agaacccatc
     2341 cacattcctg gcagcatcca accctgtggc tgcctcctgg cttgtgatgc acaagcagtc
     2401 aggattacaa ggattacaga aaatgctgga gccttctttg gaagggaaac ccctagggtg
     2461 ggagagctgt tggccgatta ctttggcgaa acagaggccc atgccttgag aaacgcactg
     2521 gctcagagct ctgatcctaa aaggcctgct ctgatctttg gctggagaga tggcctcact
     2581 ggcagaacct tcgatattag cctccatagg cacgatggaa cctccattat tgaatttgag
     2641 cccgctgccg ctgagcaggc tgataaccct ctgagactca caagacaaat cattgccagg
     2701 accaaggaac tcaagtccct ggaagaaatg gcagccaggg tgccaagata tctccaggct
     2761 atgctgggct accacagagt catgctgtat aggtttgctg atgatggaag cgggaaggtc
     2821 attggggagg caaagaggtc tgacctcgaa agcttcttgg gacaacactt ccctgccagc
     2881 ctggtccccc agcaggccag gctgctctat ctcaagaatg ccatcagggt ggtgtctgat
     2941 agcagaggga tcagctccag aatcgtccct gagcatgacg cctctggcgc tgccctggat
     3001 ttgagctttg ctcacctcag gagcatctct cctatacatt tggaatttct gaggaacatg
     3061 ggggtgtctg cctccatgag cctgagcatc attatcgatg gcaccctctg gggcctcatt
     3121 atctgccacc actatgaacc tagagctgtc cctatggcac agagagtggc tgctgagatg
     3181 ttcgctgatt ttctcagctt gcactttact gctgctcatc accaaagata agagttcgcg
     3241 tctggaacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3301 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3361 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3421 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3481 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3541 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3601 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     3661 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3721 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3781 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     3841 gaattaattc tggctaagca cttcgtggcc gtcgatcgtt taaagggagg tagtgagtcg
     3901 agatatcgat actagtgatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     3961 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4021 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4081 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4141 tatggctgat tatgatcctc tagagggaca gccccccccc aaagccccca gggatgtaat
     4201 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
     4261 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
     4321 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
     4381 ggatacgggg aaaagccata ccaatggagc tcacggggac agcccccccc caaagccccc
     4441 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     4501 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     4561 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     4621 gacacctggg gggatacggg gaaaaagaat tcgataaaag ttttgttact ttatagaaga
     4681 aattttgagt ttttgttttt tttaataaat aaataaacat aaataaattg tttgttgaat
     4741 ttattattag tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat
     4801 aaacctcgat atacagaccg ataaaacaca tgcgtcaatt ttacacatga ttatctttaa
     4861 cgtacgtcac aatatgatta tctttctagg gttaatctag ctgcgtgttc tgcagcgtgt
     4921 cgagcatctt catctgctcc atcacgctgt aaaacacatt tgcaccgcga gtctgcccgt
     4981 cctccacggg ttcaaaaacg tgaatgaacg aggcgcgctc actggccgtc gttttacaac
     5041 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     5101 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     5161 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5221 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5281 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5341 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5401 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5461 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5521 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5581 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     5641 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5701 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5761 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5821 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5881 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5941 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     6001 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     6061 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     6121 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6181 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6241 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6301 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6361 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6421 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6481 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6541 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6601 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6661 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6721 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6781 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6841 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6901 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6961 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     7021 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     7081 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     7141 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7201 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7261 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7321 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7381 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7441 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7501 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7561 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7621 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     7681 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     7741 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     7801 gcgcgcccgc cgggtaactc acggggtatc catgtccatt tctgcggcat ccagccagga
     7861 tacccgtcct cgctgacgta atatcccagc gccgcaccgc tgtcattaat ctgcacaccg
     7921 gcacggcagt tccggctgtc gccggtattg ttcgggttgc tgatgcgctt cgggctgacc
     7981 atccggaact gtgtccggaa aagccgcgac gaactggtat cccaggtggc ctgaacgaac
     8041 agttcaccgt taaaggcgtg catggccaca ccttcccgaa tcatcatggt aaacgtgcgt
     8101 tttcgctcaa cgtcaatgca gcagcagtca tcctcggcaa actctttcca tgccgcttca
     8161 acctcgcggg aaaaggcacg ggcttcttcc tccccgatgc ccagatagcg ccagcttggg
     8221 cgatgactga gccggaaaaa agacccgacg atatgatcct gatgcagcta ga
//
