LOCUS       sequence-267398-        8273 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 8273)
  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 8273)
  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-Luc
FEATURES             Location/Qualifiers
     source          1..8273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     63..79
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     63..79
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   570..604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(873..889)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(873..889)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1145..1393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1145..1165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1316..1335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2768..2821
                     /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             2828..2854
                     /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             2828..2854
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3814..4402
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3867..3887)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4285..4296)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4482..4616
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4506..4525)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4560..4579
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5395..5457
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5587..5604)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5587..5603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5596..5618)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5745..6200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5832..5851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6042..6063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6226..6330
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6331..6399,6400..7191)
                     /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(6549..6568)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7362..7950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7851..7870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8104..8121
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8238..8259
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
       61 cacaggaaac agctatgacc atgattacgc caagcgcgcc cgccgggtaa ctcacggggt
      121 atccatgtcc atttctgcgg catccagcca ggatacccgt cctcgctgac gtaatatccc
      181 agcgccgcac cgctgtcatt aatctgcaca ccggcacggc agttccggct gtcgccggta
      241 ttgttcgggt tgctgatgcg cttcgggctg accatccgga actgtgtccg gaaaagccgc
      301 gacgaactgg tatcccaggt ggcctgaacg aacagttcac cgttaaaggc gtgcatggcc
      361 acaccttccc gaatcatcat ggtaaacgtg cgttttcgct caacgtcaat gcagcagcag
      421 tcatcctcgg caaactcttt ccatgccgct tcaacctcgc gggaaaaggc acgggcttct
      481 tcctccccga tgcccagata gcgccagctt gggcgatgac tgagccggaa aaaagacccg
      541 acgatatgat cctgatgcag ctagattaac cctagaaaga tagtctgcgt aaaattgacg
      601 catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc gctgtgcatt
      661 taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat gcggtaagtg
      721 tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt atcttttacg
      781 tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc tacttacgtg
      841 ataacttatt atatatatat tttcttgtta tagatccact agttctagag ggacagcccc
      901 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
      961 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
     1021 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
     1081 tctttgagcc tgcagacacc tggggggata cggggaaaag gcctccaggg ccgccccctt
     1141 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1201 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1261 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1321 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1381 aggacgaaac accggtcaca gaatcgtcgt atgcagctcg agctgcatac gacgattctg
     1441 tgatttttgc ctccaaggcc tgcaggagtc aatgggaaaa acccattgga gccaagtaca
     1501 ctgactcaat agggactttc cattgggttt tgcccagtac ataaggtcaa tagggggtga
     1561 gtcaacagga aagtcccatt ggagccaagt acattgagtc aatagggact ttccaatggg
     1621 ttttgcccag tacataaggt caatgggagg taagccaatg ggtttttccc attactgaca
     1681 tgtatactga gtcattaggg actttccaat gggttttgcc cagtacataa ggtcaatagg
     1741 ggtgaatcaa caggaaagtc ccattggagc caagtacact gagtcaatag ggactttcca
     1801 ttgggttttg cccagtacaa aaggtcaata gggggtgagt caatgggttt ttcccattat
     1861 tggcacatac ataaggtcaa taggggtgac tagtggagaa gagcatgctt gagggctgag
     1921 tgcccctcag tgggcagaga gcacatggcc cacagtccct gagaagttgg ggggaggggt
     1981 gggcaattga actggtgcct agagaaggtg gggcttgggt aaactgggaa agtgatgtgg
     2041 tgtactggct ccaccttttt ccccagggtg ggggagaacc atatataagt gcagtagtct
     2101 ctgtgaacat tcggtttagt gaacctgcag gtcggccgcc acgaccggtg ccaccatgac
     2161 tgaatacaaa ccaactgttc gcctggcaac tcgtgatgat gttccacgtg cagttcgcac
     2221 cctggctgct gcatttgctg actaccctgc aacccgtcac actgtggacc cagaccgcca
     2281 cattgaacgt gtgactgaac tgcaggagct gttcctgacc cgtgtgggcc tggacattgg
     2341 caaagtgtgg gtggcagatg atggtgctgc tgtggcagtg tggaccaccc ctgaatctgt
     2401 tgaagctggt gcagtgtttg ctgagattgg cccacgcatg gcagaactgt ctggcagccg
     2461 cctggcagca caacagcaga tggaaggtct gctggcacca caccgcccaa aagaacctgc
     2521 ttggttcctg gcaactgtgg gtgtgagccc tgaccaccag ggtaagggcc tgggctctgc
     2581 agtggtgctg cctggtgtgg aagcagctga acgtgcaggt gtgcctgctt tcctggagac
     2641 ctcagctcca cgcaacctgc ctttctatga acgcctgggc ttcactgtga ctgctgatgt
     2701 ggaagtgcca gaaggcccac gcacttggtg catgactcgc aaaccaggtg ctagccttgg
     2761 ctccggcgag ggcaggggaa gtcttctaac atgcggggac gtggaggaaa atcccggccc
     2821 actcggctac ccctacgacg tgcccgacta cgccagcggc atggccagaa aggtggatct
     2881 gacaagctgt gacagagaac ccatccacat tcctggcagc atccaaccct gtggctgcct
     2941 cctggcttgt gatgcacaag cagtcaggat tacaaggatt acagaaaatg ctggagcctt
     3001 ctttggaagg gaaaccccta gggtgggaga gctgttggcc gattactttg gcgaaacaga
     3061 ggcccatgcc ttgagaaacg cactggctca gagctctgat cctaaaaggc ctgctctgat
     3121 ctttggctgg agagatggcc tcactggcag aaccttcgat attagcctcc ataggcacga
     3181 tggaacctcc attattgaat ttgagcccgc tgccgctgag caggctgata accctctgag
     3241 actcacaaga caaatcattg ccaggaccaa ggaactcaag tccctggaag aaatggcagc
     3301 cagggtgcca agatatctcc aggctatgct gggctaccac agagtcatgc tgtataggtt
     3361 tgctgatgat ggaagcggga aggtcattgg ggaggcaaag aggtctgacc tcgaaagctt
     3421 cttgggacaa cacttccctg ccagcctggt cccccagcag gccaggctgc tctatctcaa
     3481 gaatgccatc agggtggtgt ctgatagcag agggatcagc tccagaatcg tccctgagca
     3541 tgacgcctct ggcgctgccc tggatttgag ctttgctcac ctcaggagca tctctcctat
     3601 acatttggaa tttctgagga acatgggggt gtctgcctcc atgagcctga gcatcattat
     3661 cgatggcacc ctctggggcc tcattatctg ccaccactat gaacctagag ctgtccctat
     3721 ggcacagaga gtggctgctg agatgttcgc tgattttctc agcttgcact ttactgctgc
     3781 tcatcaccaa agataagagt tcgcgtctgg aacaatcaac ctctggatta caaaatttgt
     3841 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3901 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3961 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     4021 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     4081 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     4141 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     4201 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     4261 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     4321 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     4381 tccctttggg ccgcctcccc gcctggaatt aattctggct aagcacttcg tggccgtcga
     4441 tcgtttaaag ggaggtagtg agtcgagata tcgatactag tgatccagac atgataagat
     4501 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     4561 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     4621 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     4681 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctagag ggacagcccc
     4741 cccccaaagc ccccagggat gtaattacgt ccctcccccg ctagggggca gcagcgagcc
     4801 gcccggggct ccgctccggt ccggcgctcc ccccgcatcc ccgagccggc agcgtgcggg
     4861 gacagcccgg gcacggggaa ggtggcacgg gatcgctttc ctctgaacgc ttctcgctgc
     4921 tctttgagcc tgcagacacc tggggggata cggggaaaaa gccataccaa tggagctcac
     4981 ggggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc cgctaggggg
     5041 cagcagcgag ccgcccgggg ctccgctccg gtccggcgct ccccccgcat ccccgagccg
     5101 gcagcgtgcg gggacagccc gggcacgggg aaggtggcac gggatcgctt tcctctgaac
     5161 gcttctcgct gctctttgag cctgcagaca cctgggggga tacggggaaa aagaattcga
     5221 taaaagtttt gttactttat agaagaaatt ttgagttttt gtttttttta ataaataaat
     5281 aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa tataataaaa
     5341 cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa aacacatgcg
     5401 tcaattttac acatgattat ctttaacgta cgtcacaata tgattatctt tctagggtta
     5461 atctagctgc gtgttctgca gcgtgtcgag catcttcatc tgctccatca cgctgtaaaa
     5521 cacatttgca ccgcgagtct gcccgtcctc cacgggttca aaaacgtgaa tgaacgaggc
     5581 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     5641 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     5701 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     5761 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5821 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5881 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5941 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     6001 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6061 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6121 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     6181 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6241 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6301 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6361 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6421 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6481 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6541 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6601 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6661 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6721 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6781 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6841 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6901 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6961 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     7021 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7081 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7141 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7201 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7261 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7321 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7381 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7441 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7501 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7561 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7621 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7681 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7741 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7801 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7861 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7921 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7981 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     8041 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     8101 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     8161 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     8221 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggc
//
