LOCUS       sequence-268600-        7993 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 7993)
  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 7993)
  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
FEATURES             Location/Qualifiers
     source          1..7993
                     /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        579..827
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     579..599
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     750..769
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        936..1239
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1240..1443
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1394..1414
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2099..2152
                     /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             2159..2185
                     /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             2159..2185
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2192..2194,2195..2197,2198..2908)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2237..2258)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2498..2517)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2845..2866
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2912..2932
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2969..3557
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3022..3042)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3440..3451)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3637..3771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3661..3680)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3715..3734
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4550..4612
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4742..4759)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4742..4758)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4751..4773)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4900..5355
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4987..5006)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5197..5218
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5381..5485
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5486..5554,5555..6346)
                     /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(5704..5723)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6517..7105
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7006..7025
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7259..7276
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7393..7414
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7429..7434,7435..7452,7453..7459)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7467..7483
                     /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     7472..7494
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7491..7507
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7491..7507
                     /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 ccttcaccga gggcctattt cccatgattc
      601 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
      661 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
      721 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
      781 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg agacgctcga
      841 cccgtctcgt ttttgcctcc aaggcctgca ggcattagtt attaatagta atcaattacg
      901 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      961 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1021 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1081 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1141 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1201 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1261 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1321 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1381 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1441 gctggtttag tgaacctgca ggtcggccgc cacgaccggt gccaccatga ctgaatacaa
     1501 accaactgtt cgcctggcaa ctcgtgatga tgttccacgt gcagttcgca ccctggctgc
     1561 tgcatttgct gactaccctg caacccgtca cactgtggac ccagaccgcc acattgaacg
     1621 tgtgactgaa ctgcaggagc tgttcctgac ccgtgtgggc ctggacattg gcaaagtgtg
     1681 ggtggcagat gatggtgctg ctgtggcagt gtggaccacc cctgaatctg ttgaagctgg
     1741 tgcagtgttt gctgagattg gcccacgcat ggcagaactg tctggcagcc gcctggcagc
     1801 acaacagcag atggaaggtc tgctggcacc acaccgccca aaagaacctg cttggttcct
     1861 ggcaactgtg ggtgtgagcc ctgaccacca gggtaagggc ctgggctctg cagtggtgct
     1921 gcctggtgtg gaagcagctg aacgtgcagg tgtgcctgct ttcctggaga cctcagctcc
     1981 acgcaacctg cctttctatg aacgcctggg cttcactgtg actgctgatg tggaagtgcc
     2041 agaaggccca cgcacttggt gcatgactcg caaaccaggt gctagccttg gctccggcga
     2101 gggcagggga agtcttctaa catgcgggga cgtggaggaa aatcccggcc cactcggcta
     2161 cccctacgac gtgcccgact acgccagcgg catggtgagc aagggcgagg agctgttcac
     2221 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2281 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2341 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcacct acggcgtgca
     2401 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2461 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2521 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2581 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2641 ggtctatatc accgccgaca agcagaagaa cggcatcaag gtgaacttca agacccgcca
     2701 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2761 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2821 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2881 cactctcggc atggacgagc tgtacaaggg ccccaagaag aagcgcaagg tgggcgccta
     2941 agtacaagta agagttcgcg tctggaacaa tcaacctctg gattacaaaa tttgtgaaag
     3001 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3061 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3121 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3181 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3241 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3301 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3361 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3421 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3481 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3541 ttgggccgcc tccccgcctg gaattaattc tggctaagca cttcgtggcc gtcgatcgtt
     3601 taaagggagg tagtgagtcg agatatcgat actagtgatc cagacatgat aagatacatt
     3661 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3721 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3781 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag
     3841 taaaacctct acaaatgtgg tatggctgat tatgatcctc tagagggaca gccccccccc
     3901 aaagccccca gggatgtaat tacgtccctc ccccgctagg gggcagcagc gagccgcccg
     3961 gggctccgct ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag
     4021 cccgggcacg gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt
     4081 gagcctgcag acacctgggg ggatacgggg aaaaagccat accaatggag ctcacgggga
     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 ggaaaaagaa ttcgataaaa
     4381 gttttgttac tttatagaag aaattttgag tttttgtttt ttttaataaa taaataaaca
     4441 taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa
     4501 tatctattca aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat
     4561 tttacacatg attatcttta acgtacgtca caatatgatt atctttctag ggttaatcta
     4621 gctgcgtgtt ctgcagcgtg tcgagcatct tcatctgctc catcacgctg taaaacacat
     4681 ttgcaccgcg agtctgcccg tcctccacgg gttcaaaaac gtgaatgaac gaggcgcgct
     4741 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4801 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4861 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     4921 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4981 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     5041 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     5101 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     5161 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     5221 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5281 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5341 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5401 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5461 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5521 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5581 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5641 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5701 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     5761 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     5821 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     5881 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     5941 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6001 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6061 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6121 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6181 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6241 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6301 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6361 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6421 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6481 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6541 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6601 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6661 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6721 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6781 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6841 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6901 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6961 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7021 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7081 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7141 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7201 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7261 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     7321 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     7381 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     7441 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     7501 ctatgaccat gattacgcca agcgcgcccg ccgggtaact cacggggtat ccatgtccat
     7561 ttctgcggca tccagccagg atacccgtcc tcgctgacgt aatatcccag cgccgcaccg
     7621 ctgtcattaa tctgcacacc ggcacggcag ttccggctgt cgccggtatt gttcgggttg
     7681 ctgatgcgct tcgggctgac catccggaac tgtgtccgga aaagccgcga cgaactggta
     7741 tcccaggtgg cctgaacgaa cagttcaccg ttaaaggcgt gcatggccac accttcccga
     7801 atcatcatgg taaacgtgcg ttttcgctca acgtcaatgc agcagcagtc atcctcggca
     7861 aactctttcc atgccgcttc aacctcgcgg gaaaaggcac gggcttcttc ctccccgatg
     7921 cccagatagc gccagcttgg gcgatgactg agccggaaaa aagacccgac gatatgatcc
     7981 tgatgcagct aga
//
