LOCUS       sequence-267394-        8068 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 8068)
  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 8068)
  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
FEATURES             Location/Qualifiers
     source          1..8068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..17
                     /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     6..28
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     25..41
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     25..41
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   532..566
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(835..851)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(835..851)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1106..1354
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1106..1126
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1277..1296
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2701..2754
                     /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             2761..2787
                     /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             2761..2787
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2794..2796,2797..2799,2800..3510)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2839..2860)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3100..3119)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3447..3468
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3514..3534
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3571..4159
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3624..3644)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4042..4053)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4239..4373
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4263..4282)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4317..4336
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5152..5214
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5344..5361)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5344..5360)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5353..5375)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5502..5957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5589..5608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5799..5820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5983..6087
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6088..6156,6157..6948)
                     /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(6306..6325)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7119..7707
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7608..7627
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7861..7878
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7995..8016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8031..8036,8037..8054,8055..8061)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
       61 cccgccgggt aactcacggg gtatccatgt ccatttctgc ggcatccagc caggataccc
      121 gtcctcgctg acgtaatatc ccagcgccgc accgctgtca ttaatctgca caccggcacg
      181 gcagttccgg ctgtcgccgg tattgttcgg gttgctgatg cgcttcgggc tgaccatccg
      241 gaactgtgtc cggaaaagcc gcgacgaact ggtatcccag gtggcctgaa cgaacagttc
      301 accgttaaag gcgtgcatgg ccacaccttc ccgaatcatc atggtaaacg tgcgttttcg
      361 ctcaacgtca atgcagcagc agtcatcctc ggcaaactct ttccatgccg cttcaacctc
      421 gcgggaaaag gcacgggctt cttcctcccc gatgcccaga tagcgccagc ttgggcgatg
      481 actgagccgg aaaaaagacc cgacgatatg atcctgatgc agctagatta accctagaaa
      541 gatagtctgc gtaaaattga cgcatgcatt cttgaaatat tgctctctct ttctaaatag
      601 cgcgaatccg tcgctgtgca tttaggacat ctcagtcgcc gcttggagct cccgtgaggc
      661 gtgcttgtca atgcggtaag tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa
      721 tgacgcatga ttatctttta cgtgactttt aagatttaac tcatacgata attatattgt
      781 tatttcatgt tctacttacg tgataactta ttatatatat attttcttgt tatagatcca
      841 ctagttctag agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc
      901 cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat
      961 ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt
     1021 tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa
     1081 aggcctccag ggccgcccct tcaccgaggg cctatttccc atgattcctt catatttgca
     1141 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     1201 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     1261 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     1321 ggctttatat atcttgtgga aaggacgaaa caccgggaga cgctcgaccc gtctcgtttt
     1381 tgcctccaag gcctgcagga gtcaatggga aaaacccatt ggagccaagt acactgactc
     1441 aatagggact ttccattggg ttttgcccag tacataaggt caataggggg tgagtcaaca
     1501 ggaaagtccc attggagcca agtacattga gtcaataggg actttccaat gggttttgcc
     1561 cagtacataa ggtcaatggg aggtaagcca atgggttttt cccattactg acatgtatac
     1621 tgagtcatta gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat
     1681 caacaggaaa gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt
     1741 ttgcccagta caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcaca
     1801 tacataaggt caataggggt gactagtgga gaagagcatg cttgagggct gagtgcccct
     1861 cagtgggcag agagcacatg gcccacagtc cctgagaagt tggggggagg ggtgggcaat
     1921 tgaactggtg cctagagaag gtggggcttg ggtaaactgg gaaagtgatg tggtgtactg
     1981 gctccacctt tttccccagg gtgggggaga accatatata agtgcagtag tctctgtgaa
     2041 cattcggttt agtgaacctg caggtcggcc gccacgaccg gtgccaccat gactgaatac
     2101 aaaccaactg ttcgcctggc aactcgtgat gatgttccac gtgcagttcg caccctggct
     2161 gctgcatttg ctgactaccc tgcaacccgt cacactgtgg acccagaccg ccacattgaa
     2221 cgtgtgactg aactgcagga gctgttcctg acccgtgtgg gcctggacat tggcaaagtg
     2281 tgggtggcag atgatggtgc tgctgtggca gtgtggacca cccctgaatc tgttgaagct
     2341 ggtgcagtgt ttgctgagat tggcccacgc atggcagaac tgtctggcag ccgcctggca
     2401 gcacaacagc agatggaagg tctgctggca ccacaccgcc caaaagaacc tgcttggttc
     2461 ctggcaactg tgggtgtgag ccctgaccac cagggtaagg gcctgggctc tgcagtggtg
     2521 ctgcctggtg tggaagcagc tgaacgtgca ggtgtgcctg ctttcctgga gacctcagct
     2581 ccacgcaacc tgcctttcta tgaacgcctg ggcttcactg tgactgctga tgtggaagtg
     2641 ccagaaggcc cacgcacttg gtgcatgact cgcaaaccag gtgctagcct tggctccggc
     2701 gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccactcggc
     2761 tacccctacg acgtgcccga ctacgccagc ggcatggtga gcaagggcga ggagctgttc
     2821 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2881 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2941 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
     3001 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3061 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3121 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3181 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3241 aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc
     3301 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3361 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3421 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3481 atcactctcg gcatggacga gctgtacaag ggccccaaga agaagcgcaa ggtgggcgcc
     3541 taagtacaag taagagttcg cgtctggaac aatcaacctc tggattacaa aatttgtgaa
     3601 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3661 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3721 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3781 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3841 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3901 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3961 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg
     4021 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4081 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4141 ctttgggccg cctccccgcc tggaattaat tctggctaag cacttcgtgg ccgtcgatcg
     4201 tttaaaggga ggtagtgagt cgagatatcg atactagtga tccagacatg ataagataca
     4261 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4321 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4381 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     4441 agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctagaggga cagccccccc
     4501 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     4561 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     4621 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     4681 ttgagcctgc agacacctgg ggggatacgg ggaaaaagcc ataccaatgg agctcacggg
     4741 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag
     4801 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca
     4861 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct
     4921 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag aattcgataa
     4981 aagttttgtt actttataga agaaattttg agtttttgtt ttttttaata aataaataaa
     5041 cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt
     5101 aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca
     5161 attttacaca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc
     5221 tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc tgtaaaacac
     5281 atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga acgaggcgcg
     5341 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5401 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5461 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     5521 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5581 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5641 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5701 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5761 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5821 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5881 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5941 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6001 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6061 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6121 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6181 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6241 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6301 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6361 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6421 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6481 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6541 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6601 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6661 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6721 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6781 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6841 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6901 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6961 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7021 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7081 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7141 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7201 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7261 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7321 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7381 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7441 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7501 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7561 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7621 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7681 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7741 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7801 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7861 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7921 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     7981 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     8041 atgcttccgg ctcgtatgtt gtgtggaa
//
