LOCUS       sequence-256346-        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        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        937..1240
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1241..1444
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1395..1415
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2100..2153
                     /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             2160..2186
                     /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             2160..2186
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2193..2195,2196..2198,2199..2909)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2238..2259)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2499..2518)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2846..2867
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2913..2933
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2970..3558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3023..3043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3441..3452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3638..3772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3662..3681)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3716..3735
                     /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 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 aggcattagt tattaatagt aatcaattac
      901 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      961 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1021 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1081 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     1141 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     1201 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     1261 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     1321 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     1381 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     1441 agctggttta gtgaacctgc aggtcggccg ccacgaccgg tgccaccatg actgaataca
     1501 aaccaactgt tcgcctggca actcgtgatg atgttccacg tgcagttcgc accctggctg
     1561 ctgcatttgc tgactaccct gcaacccgtc acactgtgga cccagaccgc cacattgaac
     1621 gtgtgactga actgcaggag ctgttcctga cccgtgtggg cctggacatt ggcaaagtgt
     1681 gggtggcaga tgatggtgct gctgtggcag tgtggaccac ccctgaatct gttgaagctg
     1741 gtgcagtgtt tgctgagatt ggcccacgca tggcagaact gtctggcagc cgcctggcag
     1801 cacaacagca gatggaaggt ctgctggcac cacaccgccc aaaagaacct gcttggttcc
     1861 tggcaactgt gggtgtgagc cctgaccacc agggtaaggg cctgggctct gcagtggtgc
     1921 tgcctggtgt ggaagcagct gaacgtgcag gtgtgcctgc tttcctggag acctcagctc
     1981 cacgcaacct gcctttctat gaacgcctgg gcttcactgt gactgctgat gtggaagtgc
     2041 cagaaggccc acgcacttgg tgcatgactc gcaaaccagg tgctagcctt ggctccggcg
     2101 agggcagggg aagtcttcta acatgcgggg acgtggagga aaatcccggc ccactcggct
     2161 acccctacga cgtgcccgac tacgccagcg gcatggtgag caagggcgag gagctgttca
     2221 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2281 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     2341 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcacc tacggcgtgc
     2401 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2461 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2521 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2581 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     2641 aggtctatat caccgccgac aagcagaaga acggcatcaa ggtgaacttc aagacccgcc
     2701 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2761 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2821 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2881 tcactctcgg catggacgag ctgtacaagg gccccaagaa gaagcgcaag gtgggcgcct
     2941 aagtacaagt aagagttcgc gtctggaaca atcaacctct ggattacaaa atttgtgaaa
     3001 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     3061 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     3121 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     3181 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     3241 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     3301 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     3361 ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     3421 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     3481 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     3541 tttgggccgc ctccccgcct ggaattaatt ctggctaagc acttcgtggc cgtcgatcgt
     3601 ttaaagggag gtagtgagtc gagatatcga tactagtgat ccagacatga taagatacat
     3661 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3721 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3781 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     3841 gtaaaacctc tacaaatgtg gtatggctga ttatgatcct ctagagggac agcccccccc
     3901 caaagccccc agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc
     3961 ggggctccgc tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca
     4021 gcccgggcac ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt
     4081 tgagcctgca gacacctggg gggatacggg gaaaagccat 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 tgtccttcta 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
//
