LOCUS       sequence-267395-        8097 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 8097)
  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 8097)
  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-PnGW-Luc
FEATURES             Location/Qualifiers
     source          1..8097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   491..525
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(794..810)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(794..810)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1066..1314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1066..1086
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1237..1256
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2689..2742
                     /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             2749..2775
                     /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             2749..2775
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2782..2784,2785..2787,2788..3498)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2827..2848)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3088..3107)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3435..3456
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3502..3522
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3559..4147
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3612..3632)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4030..4041)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4227..4361
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4251..4270)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4305..4324
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5140..5202
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5332..5349)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5332..5348)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5341..5363)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5490..5945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5577..5596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5787..5808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5971..6075
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6076..6144,6145..6936)
                     /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(6294..6313)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7107..7695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7596..7615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7849..7866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7983..8004
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8019..8024,8025..8042,8043..8049)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8057..8073
                     /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     8062..8084
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8081..8097
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8081..8097
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 catgattacg ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg
       61 gcatccagcc aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat
      121 taatctgcac accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc
      181 gcttcgggct gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg
      241 tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca
      301 tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt
      361 tccatgccgc ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat
      421 agcgccagct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca
      481 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt
      541 gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg
      601 cttggagctc ccgtgaggcg tgcttgtcaa tgcggtaagt gtcactgatt ttgaactata
      661 acgaccgcgt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact
      721 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata
      781 ttttcttgtt atagatccac tagttctaga gggacagccc ccccccaaag cccccaggga
      841 tgtaattacg tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg
      901 tccggcgctc cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga
      961 aggtggcacg ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac
     1021 ctggggggat acggggaaaa ggcctccagg gccgccccct tcaccgaggg cctatttccc
     1081 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     1141 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     1201 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     1261 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtcac
     1321 agaatcgtcg tatgcagctc gagctgcata cgacgattct gtgatttttg cctccaaggc
     1381 ctgcaggagt caatgggaaa aacccattgg agccaagtac actgactcaa tagggacttt
     1441 ccattgggtt ttgcccagta cataaggtca atagggggtg agtcaacagg aaagtcccat
     1501 tggagccaag tacattgagt caatagggac tttccaatgg gttttgccca gtacataagg
     1561 tcaatgggag gtaagccaat gggtttttcc cattactgac atgtatactg agtcattagg
     1621 gactttccaa tgggttttgc ccagtacata aggtcaatag gggtgaatca acaggaaagt
     1681 cccattggag ccaagtacac tgagtcaata gggactttcc attgggtttt gcccagtaca
     1741 aaaggtcaat agggggtgag tcaatgggtt tttcccatta ttggcacata cataaggtca
     1801 ataggggtga ctagtggaga agagcatgct tgagggctga gtgcccctca gtgggcagag
     1861 agcacatggc ccacagtccc tgagaagttg gggggagggg tgggcaattg aactggtgcc
     1921 tagagaaggt ggggcttggg taaactggga aagtgatgtg gtgtactggc tccacctttt
     1981 tccccagggt gggggagaac catatataag tgcagtagtc tctgtgaaca ttcggtttag
     2041 tgaacctgca ggtcggccgc cacgaccggt gccaccatga ctgaatacaa accaactgtt
     2101 cgcctggcaa ctcgtgatga tgttccacgt gcagttcgca ccctggctgc tgcatttgct
     2161 gactaccctg caacccgtca cactgtggac ccagaccgcc acattgaacg tgtgactgaa
     2221 ctgcaggagc tgttcctgac ccgtgtgggc ctggacattg gcaaagtgtg ggtggcagat
     2281 gatggtgctg ctgtggcagt gtggaccacc cctgaatctg ttgaagctgg tgcagtgttt
     2341 gctgagattg gcccacgcat ggcagaactg tctggcagcc gcctggcagc acaacagcag
     2401 atggaaggtc tgctggcacc acaccgccca aaagaacctg cttggttcct ggcaactgtg
     2461 ggtgtgagcc ctgaccacca gggtaagggc ctgggctctg cagtggtgct gcctggtgtg
     2521 gaagcagctg aacgtgcagg tgtgcctgct ttcctggaga cctcagctcc acgcaacctg
     2581 cctttctatg aacgcctggg cttcactgtg actgctgatg tggaagtgcc agaaggccca
     2641 cgcacttggt gcatgactcg caaaccaggt gctagccttg gctccggcga gggcagggga
     2701 agtcttctaa catgcgggga cgtggaggaa aatcccggcc cactcggcta cccctacgac
     2761 gtgcccgact acgccagcgg catggtgagc aagggcgagg agctgttcac cggggtggtg
     2821 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2881 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2941 ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca gtgcttcgcc
     3001 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     3061 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     3121 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     3181 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa ggtctatatc
     3241 accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca caacatcgag
     3301 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3361 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     3421 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3481 atggacgagc tgtacaaggg ccccaagaag aagcgcaagg tgggcgccta agtacaagta
     3541 agagttcgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3601 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3661 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3721 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3781 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3841 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3901 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     3961 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     4021 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     4081 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     4141 tccccgcctg gaattaattc tggctaagca cttcgtggcc gtcgatcgtt taaagggagg
     4201 tagtgagtcg agatatcgat actagtgatc cagacatgat aagatacatt gatgagtttg
     4261 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     4321 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4381 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct
     4441 acaaatgtgg tatggctgat tatgatcctc tagagggaca gccccccccc aaagccccca
     4501 gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct
     4561 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg
     4621 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag
     4681 acacctgggg ggatacgggg aaaaagccat accaatggag ctcacgggga cagccccccc
     4741 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     4801 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     4861 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     4921 ttgagcctgc agacacctgg ggggatacgg ggaaaaagaa ttcgataaaa gttttgttac
     4981 tttatagaag aaattttgag tttttgtttt ttttaataaa taaataaaca taaataaatt
     5041 gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa tatctattca
     5101 aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat tttacacatg
     5161 attatcttta acgtacgtca caatatgatt atctttctag ggttaatcta gctgcgtgtt
     5221 ctgcagcgtg tcgagcatct tcatctgctc catcacgctg taaaacacat ttgcaccgcg
     5281 agtctgcccg tcctccacgg gttcaaaaac gtgaatgaac gaggcgcgct cactggccgt
     5341 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     5401 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     5461 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     5521 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     5581 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     5641 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     5701 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     5761 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     5821 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     5881 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     5941 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     6001 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     6061 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     6121 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     6181 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     6241 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     6301 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     6361 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     6421 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     6481 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6541 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6601 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6661 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6721 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6781 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6841 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6901 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6961 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     7021 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7081 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     7141 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7201 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     7261 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7321 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7381 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7441 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7501 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7561 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7621 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7681 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7741 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7801 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7861 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7921 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7981 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     8041 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgac
//
