LOCUS       sequence-256339-        8096 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 8096)
  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 8096)
  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..8096
                     /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"
     CDS             join(2296..2298,2299..2301,2302..3012)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2341..2362)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2602..2621)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2949..2970
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3016..3036
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3073..3661
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3126..3146)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3544..3555)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3741..3875
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3765..3784)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3819..3838
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4653..4715
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4845..4862)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4845..4861)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4854..4876)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5003..5458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5090..5109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5300..5321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5484..5588
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5589..5657,5658..6449)
                     /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(5807..5826)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6620..7208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7109..7128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7362..7379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7496..7517
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7532..7537,7538..7555,7556..7562)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7570..7586
                     /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     7575..7597
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7594..7610
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7594..7610
                     /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 gcggcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2341 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2401 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     2461 gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt cgcccgctac
     2521 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     2581 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     2641 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2701 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaaggtcta tatcaccgcc
     2761 gacaagcaga agaacggcat caaggtgaac ttcaagaccc gccacaacat cgaggacggc
     2821 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2881 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2941 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3001 gagctgtaca agggccccaa gaagaagcgc aaggtgggcg cctaagtaca agtaagagtt
     3061 cgcgtctgga acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3121 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3181 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3241 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3301 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3361 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3421 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt
     3481 ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3541 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3601 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     3661 cctggaatta attctggcta agcacttcgt ggccgtcgat cgtttaaagg gaggtagtga
     3721 gtcgagatat cgatactagt gatccagaca tgataagata cattgatgag tttggacaaa
     3781 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3841 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3901 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     3961 gtggtatggc tgattatgat cctctagagg gacagccccc ccccaaagcc cccagggatg
     4021 taattacgtc cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc
     4081 cggcgctccc cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag
     4141 gtggcacggg atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct
     4201 ggggggatac ggggaaaagc cataccaatg gagctcacgg ggacagcccc cccccaaagc
     4261 ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc gcccggggct
     4321 ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg gacagcccgg
     4381 gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc tctttgagcc
     4441 tgcagacacc tggggggata cggggaaaaa gaattcgata aaagttttgt tactttatag
     4501 aagaaatttt gagtttttgt tttttttaat aaataaataa acataaataa attgtttgtt
     4561 gaatttatta ttagtatgta agtgtaaata taataaaact taatatctat tcaaattaat
     4621 aaataaacct cgatatacag accgataaaa cacatgcgtc aattttacac atgattatct
     4681 ttaacgtacg tcacaatatg attatctttc tagggttaat ctagctgcgt gttctgcagc
     4741 gtgtcgagca tcttcatctg ctccatcacg ctgtaaaaca catttgcacc gcgagtctgc
     4801 ccgtcctcca cgggttcaaa aacgtgaatg aacgaggcgc gctcactggc cgtcgtttta
     4861 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     4921 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     4981 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     5041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     5101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     5161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     5221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     5341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     5401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     5461 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5521 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5581 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5641 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5701 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5761 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5821 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5881 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5941 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     6001 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     6061 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     6121 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6181 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6241 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6301 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6361 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6421 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6481 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6541 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6601 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6661 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6721 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     6781 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6841 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6901 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6961 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7021 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7081 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7141 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7201 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7261 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7321 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7381 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7441 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7501 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     7561 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     7621 ccaagcgcgc ccgccgggta actcacgggg tatccatgtc catttctgcg gcatccagcc
     7681 aggatacccg tcctcgctga cgtaatatcc cagcgccgca ccgctgtcat taatctgcac
     7741 accggcacgg cagttccggc tgtcgccggt attgttcggg ttgctgatgc gcttcgggct
     7801 gaccatccgg aactgtgtcc ggaaaagccg cgacgaactg gtatcccagg tggcctgaac
     7861 gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca tggtaaacgt
     7921 gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt tccatgccgc
     7981 ttcaacctcg cgggaaaagg cacgggcttc ttcctccccg atgcccagat agcgccagct
     8041 tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca gctaga
//
