LOCUS       sequence-267540-        8021 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 8021)
  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 8021)
  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-CMV-Luc
FEATURES             Location/Qualifiers
     source          1..8021
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    8..24
                     /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     13..35
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     32..48
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     32..48
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   539..573
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(842..858)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(842..858)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1113..1361
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1113..1133
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1284..1303
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        1498..1801
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1802..2005
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1956..1976
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2661..2714
                     /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             2721..2747
                     /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             2721..2747
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2754..2756,2757..2759,2760..3470)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2799..2820)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3060..3079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3407..3428
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3474..3494
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3531..4119
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3584..3604)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4002..4013)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4199..4333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4223..4242)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4277..4296
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5112..5174
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5304..5321)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5304..5320)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5313..5335)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5462..5917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5549..5568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5759..5780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5943..6047
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6048..6116,6117..6908)
                     /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(6266..6285)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7079..7667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7568..7587
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7821..7838
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7955..7976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7991..7996,7997..8014,8015..8021)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
       61 aagcgcgccc gccgggtaac tcacggggta tccatgtcca tttctgcggc atccagccag
      121 gatacccgtc ctcgctgacg taatatccca gcgccgcacc gctgtcatta atctgcacac
      181 cggcacggca gttccggctg tcgccggtat tgttcgggtt gctgatgcgc ttcgggctga
      241 ccatccggaa ctgtgtccgg aaaagccgcg acgaactggt atcccaggtg gcctgaacga
      301 acagttcacc gttaaaggcg tgcatggcca caccttcccg aatcatcatg gtaaacgtgc
      361 gttttcgctc aacgtcaatg cagcagcagt catcctcggc aaactctttc catgccgctt
      421 caacctcgcg ggaaaaggca cgggcttctt cctccccgat gcccagatag cgccagcttg
      481 ggcgatgact gagccggaaa aaagacccga cgatatgatc ctgatgcagc tagattaacc
      541 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc
      601 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc
      661 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga
      721 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt
      781 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat
      841 agatccacta gttctagagg gacagccccc ccccaaagcc cccagggatg taattacgtc
      901 cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc
      961 cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg
     1021 atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac
     1081 ggggaaaagg cctccagggc cgccccttca ccgagggcct atttcccatg attccttcat
     1141 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     1201 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1261 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1321 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggtcacaga atcgtcgtat
     1381 gcagctcgag ctgcatacga cgattctgtg atttttgcct ccaaggcctg caggcattag
     1441 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     1501 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     1561 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     1621 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     1681 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     1741 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     1801 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     1861 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     1921 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     1981 gtgggaggtc tatataagca gagctggttt agtgaacctg caggtcggcc gccacgaccg
     2041 gtgccaccat gactgaatac aaaccaactg ttcgcctggc aactcgtgat gatgttccac
     2101 gtgcagttcg caccctggct gctgcatttg ctgactaccc tgcaacccgt cacactgtgg
     2161 acccagaccg ccacattgaa cgtgtgactg aactgcagga gctgttcctg acccgtgtgg
     2221 gcctggacat tggcaaagtg tgggtggcag atgatggtgc tgctgtggca gtgtggacca
     2281 cccctgaatc tgttgaagct ggtgcagtgt ttgctgagat tggcccacgc atggcagaac
     2341 tgtctggcag ccgcctggca gcacaacagc agatggaagg tctgctggca ccacaccgcc
     2401 caaaagaacc tgcttggttc ctggcaactg tgggtgtgag ccctgaccac cagggtaagg
     2461 gcctgggctc tgcagtggtg ctgcctggtg tggaagcagc tgaacgtgca ggtgtgcctg
     2521 ctttcctgga gacctcagct ccacgcaacc tgcctttcta tgaacgcctg ggcttcactg
     2581 tgactgctga tgtggaagtg ccagaaggcc cacgcacttg gtgcatgact cgcaaaccag
     2641 gtgctagcct tggctccggc gagggcaggg gaagtcttct aacatgcggg gacgtggagg
     2701 aaaatcccgg cccactcggc tacccctacg acgtgcccga ctacgccagc ggcatggtga
     2761 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2821 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2881 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2941 ccaccttcac ctacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     3001 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     3061 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     3121 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     3181 agtacaacta caacagccac aaggtctata tcaccgccga caagcagaag aacggcatca
     3241 aggtgaactt caagacccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     3301 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     3361 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     3421 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggccccaaga
     3481 agaagcgcaa ggtgggcgcc taagtacaag taagagttcg cgtctggaac aatcaacctc
     3541 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3601 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3661 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3721 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3781 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3841 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3901 acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg
     3961 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     4021 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     4081 ctcagacgag tcggatctcc ctttgggccg cctccccgcc tggaattaat tctggctaag
     4141 cacttcgtgg ccgtcgatcg tttaaaggga ggtagtgagt cgagatatcg atactagtga
     4201 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     4261 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     4321 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     4381 gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc
     4441 tctagaggga cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta
     4501 gggggcagca gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg
     4561 agccggcagc gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc
     4621 tgaacgcttc tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaaagcc
     4681 ataccaatgg agctcacggg gacagccccc ccccaaagcc cccagggatg taattacgtc
     4741 cctcccccgc tagggggcag cagcgagccg cccggggctc cgctccggtc cggcgctccc
     4801 cccgcatccc cgagccggca gcgtgcgggg acagcccggg cacggggaag gtggcacggg
     4861 atcgctttcc tctgaacgct tctcgctgct ctttgagcct gcagacacct ggggggatac
     4921 ggggaaaaag aattcgataa aagttttgtt actttataga agaaattttg agtttttgtt
     4981 ttttttaata aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa
     5041 gtgtaaatat aataaaactt aatatctatt caaattaata aataaacctc gatatacaga
     5101 ccgataaaac acatgcgtca attttacaca tgattatctt taacgtacgt cacaatatga
     5161 ttatctttct agggttaatc tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc
     5221 tccatcacgc tgtaaaacac atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa
     5281 acgtgaatga acgaggcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     5341 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     5401 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     5461 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     5521 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     5581 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     5641 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     5701 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     5761 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     5821 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     5881 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     5941 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6001 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     6061 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     6121 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     6181 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     6241 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     6301 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     6361 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     6421 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     6481 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     6541 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6601 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6661 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6721 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6781 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6841 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6901 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6961 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     7021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     7081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     7141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     7201 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     7261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     7321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     7381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     7441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     7501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     7561 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     7621 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     7681 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     7741 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     7801 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     7861 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     7921 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     7981 caccccaggc tttacacttt atgcttccgg ctcgtatgtt g
//
