LOCUS       sequence-256342-        8244 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 8244)
  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 8244)
  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
FEATURES             Location/Qualifiers
     source          1..8244
                     /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             2175..2228
                     /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             2235..2261
                     /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             2235..2261
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3221..3809
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3274..3294)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3692..3703)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3889..4023
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3913..3932)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3967..3986
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4801..4863
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4993..5010)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4993..5009)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5002..5024)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5151..5606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5238..5257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5448..5469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5632..5736
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5737..5805,5806..6597)
                     /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(5955..5974)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6768..7356
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7257..7276
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7510..7527
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7644..7665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7680..7685,7686..7703,7704..7710)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7718..7734
                     /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     7723..7745
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7742..7758
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7742..7758
                     /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 gagacgctcg
      841 acccgtctcg tttttgcctc caaggcctgc aggagtcaat gggaaaaacc cattggagcc
      901 aagtacactg actcaatagg gactttccat tgggttttgc ccagtacata aggtcaatag
      961 ggggtgagtc aacaggaaag tcccattgga gccaagtaca ttgagtcaat agggactttc
     1021 caatgggttt tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt
     1081 actgacatgt atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt
     1141 caataggggt gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga
     1201 ctttccattg ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc
     1261 ccattattgg cacatacata aggtcaatag gggtgactag tggagaagag catgcttgag
     1321 ggctgagtgc ccctcagtgg gcagagagca catggcccac agtccctgag aagttggggg
     1381 gaggggtggg caattgaact ggtgcctaga gaaggtgggg cttgggtaaa ctgggaaagt
     1441 gatgtggtgt actggctcca cctttttccc cagggtgggg gagaaccata tataagtgca
     1501 gtagtctctg tgaacattcg gtttagtgaa cctgcaggtc ggccgccacg accggtgcca
     1561 ccatgactga atacaaacca actgttcgcc tggcaactcg tgatgatgtt ccacgtgcag
     1621 ttcgcaccct ggctgctgca tttgctgact accctgcaac ccgtcacact gtggacccag
     1681 accgccacat tgaacgtgtg actgaactgc aggagctgtt cctgacccgt gtgggcctgg
     1741 acattggcaa agtgtgggtg gcagatgatg gtgctgctgt ggcagtgtgg accacccctg
     1801 aatctgttga agctggtgca gtgtttgctg agattggccc acgcatggca gaactgtctg
     1861 gcagccgcct ggcagcacaa cagcagatgg aaggtctgct ggcaccacac cgcccaaaag
     1921 aacctgcttg gttcctggca actgtgggtg tgagccctga ccaccagggt aagggcctgg
     1981 gctctgcagt ggtgctgcct ggtgtggaag cagctgaacg tgcaggtgtg cctgctttcc
     2041 tggagacctc agctccacgc aacctgcctt tctatgaacg cctgggcttc actgtgactg
     2101 ctgatgtgga agtgccagaa ggcccacgca cttggtgcat gactcgcaaa ccaggtgcta
     2161 gccttggctc cggcgagggc aggggaagtc ttctaacatg cggggacgtg gaggaaaatc
     2221 ccggcccact cggctacccc tacgacgtgc ccgactacgc cagcggcatg gccagaaagg
     2281 tggatctgac aagctgtgac agagaaccca tccacattcc tggcagcatc caaccctgtg
     2341 gctgcctcct ggcttgtgat gcacaagcag tcaggattac aaggattaca gaaaatgctg
     2401 gagccttctt tggaagggaa acccctaggg tgggagagct gttggccgat tactttggcg
     2461 aaacagaggc ccatgccttg agaaacgcac tggctcagag ctctgatcct aaaaggcctg
     2521 ctctgatctt tggctggaga gatggcctca ctggcagaac cttcgatatt agcctccata
     2581 ggcacgatgg aacctccatt attgaatttg agcccgctgc cgctgagcag gctgataacc
     2641 ctctgagact cacaagacaa atcattgcca ggaccaagga actcaagtcc ctggaagaaa
     2701 tggcagccag ggtgccaaga tatctccagg ctatgctggg ctaccacaga gtcatgctgt
     2761 ataggtttgc tgatgatgga agcgggaagg tcattgggga ggcaaagagg tctgacctcg
     2821 aaagcttctt gggacaacac ttccctgcca gcctggtccc ccagcaggcc aggctgctct
     2881 atctcaagaa tgccatcagg gtggtgtctg atagcagagg gatcagctcc agaatcgtcc
     2941 ctgagcatga cgcctctggc gctgccctgg atttgagctt tgctcacctc aggagcatct
     3001 ctcctataca tttggaattt ctgaggaaca tgggggtgtc tgcctccatg agcctgagca
     3061 tcattatcga tggcaccctc tggggcctca ttatctgcca ccactatgaa cctagagctg
     3121 tccctatggc acagagagtg gctgctgaga tgttcgctga ttttctcagc ttgcacttta
     3181 ctgctgctca tcaccaaaga taagagttcg cgtctggaac aatcaacctc tggattacaa
     3241 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3301 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3361 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3421 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3481 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     3541 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     3601 ggtgttgtcg gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat
     3661 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     3721 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     3781 tcggatctcc ctttgggccg cctccccgcc tggaattaat tctggctaag cacttcgtgg
     3841 ccgtcgatcg tttaaaggga ggtagtgagt cgagatatcg atactagtga tccagacatg
     3901 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3961 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     4021 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     4081 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctagaggga
     4141 cagccccccc ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca
     4201 gcgagccgcc cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc
     4261 gtgcggggac agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc
     4321 tcgctgctct ttgagcctgc agacacctgg ggggatacgg ggaaaagcca taccaatgga
     4381 gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct
     4441 agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc
     4501 gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct
     4561 ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaaaga
     4621 attcgataaa agttttgtta ctttatagaa gaaattttga gtttttgttt tttttaataa
     4681 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     4741 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     4801 catgcgtcaa ttttacacat gattatcttt aacgtacgtc acaatatgat tatctttcta
     4861 gggttaatct agctgcgtgt tctgcagcgt gtcgagcatc ttcatctgct ccatcacgct
     4921 gtaaaacaca tttgcaccgc gagtctgccc gtcctccacg ggttcaaaaa cgtgaatgaa
     4981 cgaggcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     5041 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5101 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     5161 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     5221 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5281 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5341 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     5401 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5461 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     5521 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5581 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     5641 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5701 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5761 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5821 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5881 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5941 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     6001 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     6061 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     6121 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     6181 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     6241 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     6301 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     6361 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     6421 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     6481 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6541 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     6601 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6661 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6721 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6781 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6841 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6901 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6961 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     7021 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     7081 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     7141 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7201 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7261 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7321 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7381 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7441 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     7501 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     7561 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     7621 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     7681 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     7741 acaggaaaca gctatgacca tgattacgcc aagcgcgccc gccgggtaac tcacggggta
     7801 tccatgtcca tttctgcggc atccagccag gatacccgtc ctcgctgacg taatatccca
     7861 gcgccgcacc gctgtcatta atctgcacac cggcacggca gttccggctg tcgccggtat
     7921 tgttcgggtt gctgatgcgc ttcgggctga ccatccggaa ctgtgtccgg aaaagccgcg
     7981 acgaactggt atcccaggtg gcctgaacga acagttcacc gttaaaggcg tgcatggcca
     8041 caccttcccg aatcatcatg gtaaacgtgc gttttcgctc aacgtcaatg cagcagcagt
     8101 catcctcggc aaactctttc catgccgctt caacctcgcg ggaaaaggca cgggcttctt
     8161 cctccccgat gcccagatag cgccagcttg ggcgatgact gagccggaaa aaagacccga
     8221 cgatatgatc ctgatgcagc taga
//
