LOCUS       sequence-256347-        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"
     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"
     enhancer        965..1268
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1269..1472
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1423..1443
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2128..2181
                     /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             2188..2214
                     /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             2188..2214
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2221..2223,2224..2226,2227..2937)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2266..2287)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2527..2546)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2874..2895
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2941..2961
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2998..3586
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3051..3071)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3469..3480)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3666..3800
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3690..3709)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3744..3763
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4578..4640
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4770..4787)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4770..4786)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4779..4801)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4928..5383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5015..5034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5225..5246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5409..5513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5514..5582,5583..6374)
                     /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(5732..5751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6545..7133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7034..7053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7287..7304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7421..7442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7457..7462,7463..7480,7481..7487)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7495..7511
                     /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     7500..7522
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7519..7535
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7519..7535
                     /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 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      961 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1021 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1081 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1141 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1201 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1261 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     1321 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     1381 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1441 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaacctgcag gtcggccgcc
     1501 acgaccggtg ccaccatgac tgaatacaaa ccaactgttc gcctggcaac tcgtgatgat
     1561 gttccacgtg cagttcgcac cctggctgct gcatttgctg actaccctgc aacccgtcac
     1621 actgtggacc cagaccgcca cattgaacgt gtgactgaac tgcaggagct gttcctgacc
     1681 cgtgtgggcc tggacattgg caaagtgtgg gtggcagatg atggtgctgc tgtggcagtg
     1741 tggaccaccc ctgaatctgt tgaagctggt gcagtgtttg ctgagattgg cccacgcatg
     1801 gcagaactgt ctggcagccg cctggcagca caacagcaga tggaaggtct gctggcacca
     1861 caccgcccaa aagaacctgc ttggttcctg gcaactgtgg gtgtgagccc tgaccaccag
     1921 ggtaagggcc tgggctctgc agtggtgctg cctggtgtgg aagcagctga acgtgcaggt
     1981 gtgcctgctt tcctggagac ctcagctcca cgcaacctgc ctttctatga acgcctgggc
     2041 ttcactgtga ctgctgatgt ggaagtgcca gaaggcccac gcacttggtg catgactcgc
     2101 aaaccaggtg ctagccttgg ctccggcgag ggcaggggaa gtcttctaac atgcggggac
     2161 gtggaggaaa atcccggccc actcggctac ccctacgacg tgcccgacta cgccagcggc
     2221 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2281 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2341 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2401 ctcgtgacca ccttcaccta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     2461 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2521 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2581 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2641 aagctggagt acaactacaa cagccacaag gtctatatca ccgccgacaa gcagaagaac
     2701 ggcatcaagg tgaacttcaa gacccgccac aacatcgagg acggcagcgt gcagctcgcc
     2761 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     2821 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     2881 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc
     2941 cccaagaaga agcgcaaggt gggcgcctaa gtacaagtaa gagttcgcgt ctggaacaat
     3001 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     3061 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     3121 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     3181 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3241 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3301 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3361 ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc
     3421 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3481 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3541 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattaattct
     3601 ggctaagcac ttcgtggccg tcgatcgttt aaagggaggt agtgagtcga gatatcgata
     3661 ctagtgatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     3721 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     3781 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     3841 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt
     3901 atgatcctct agagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc
     3961 cccgctaggg ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc
     4021 atccccgagc cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc
     4081 tttcctctga acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga
     4141 aaagccatac caatggagct cacggggaca gccccccccc aaagccccca gggatgtaat
     4201 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc
     4261 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg
     4321 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg
     4381 ggatacgggg aaaaagaatt cgataaaagt tttgttactt tatagaagaa attttgagtt
     4441 tttgtttttt ttaataaata aataaacata aataaattgt ttgttgaatt tattattagt
     4501 atgtaagtgt aaatataata aaacttaata tctattcaaa ttaataaata aacctcgata
     4561 tacagaccga taaaacacat gcgtcaattt tacacatgat tatctttaac gtacgtcaca
     4621 atatgattat ctttctaggg ttaatctagc tgcgtgttct gcagcgtgtc gagcatcttc
     4681 atctgctcca tcacgctgta aaacacattt gcaccgcgag tctgcccgtc ctccacgggt
     4741 tcaaaaacgt gaatgaacga ggcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     4801 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     4861 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4921 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     5041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     5101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     5161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     5221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     5281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     5341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5701 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6361 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6421 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6481 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6541 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6601 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6661 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6721 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6781 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6841 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6901 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6961 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     7021 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     7081 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     7141 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     7201 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7261 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     7321 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7381 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     7441 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     7501 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcccgcc
     7561 gggtaactca cggggtatcc atgtccattt ctgcggcatc cagccaggat acccgtcctc
     7621 gctgacgtaa tatcccagcg ccgcaccgct gtcattaatc tgcacaccgg cacggcagtt
     7681 ccggctgtcg ccggtattgt tcgggttgct gatgcgcttc gggctgacca tccggaactg
     7741 tgtccggaaa agccgcgacg aactggtatc ccaggtggcc tgaacgaaca gttcaccgtt
     7801 aaaggcgtgc atggccacac cttcccgaat catcatggta aacgtgcgtt ttcgctcaac
     7861 gtcaatgcag cagcagtcat cctcggcaaa ctctttccat gccgcttcaa cctcgcggga
     7921 aaaggcacgg gcttcttcct ccccgatgcc cagatagcgc cagcttgggc gatgactgag
     7981 ccggaaaaaa gacccgacga tatgatcctg atgcagctag a
//
