LOCUS       sequence-267397-        8245 bp    DNA     circular SYN 15-APR-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 8245)
  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 8245)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..8245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(15..20,21..38,39..45)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    53..69
                     /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     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   584..618
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(887..903)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(887..903)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        1159..1407
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1159..1179
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1330..1349
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             2754..2807
                     /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             2814..2840
                     /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             2814..2840
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    3800..4388
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3853..3873)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4271..4282)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4468..4602
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4492..4511)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4546..4565
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5381..5443
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5573..5590)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5573..5589)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5582..5604)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5731..6186
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5818..5837)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6028..6049
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6212..6316
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6317..6385,6386..7177)
                     /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(6535..6554)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7348..7936
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7837..7856
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8090..8107
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8224..8245
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcccgccgg
      121 gtaactcacg gggtatccat gtccatttct gcggcatcca gccaggatac ccgtcctcgc
      181 tgacgtaata tcccagcgcc gcaccgctgt cattaatctg cacaccggca cggcagttcc
      241 ggctgtcgcc ggtattgttc gggttgctga tgcgcttcgg gctgaccatc cggaactgtg
      301 tccggaaaag ccgcgacgaa ctggtatccc aggtggcctg aacgaacagt tcaccgttaa
      361 aggcgtgcat ggccacacct tcccgaatca tcatggtaaa cgtgcgtttt cgctcaacgt
      421 caatgcagca gcagtcatcc tcggcaaact ctttccatgc cgcttcaacc tcgcgggaaa
      481 aggcacgggc ttcttcctcc ccgatgccca gatagcgcca gcttgggcga tgactgagcc
      541 ggaaaaaaga cccgacgata tgatcctgat gcagctagat taaccctaga aagatagtct
      601 gcgtaaaatt gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc
      661 cgtcgctgtg catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt
      721 caatgcggta agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat
      781 gattatcttt tacgtgactt ttaagattta actcatacga taattatatt gttatttcat
      841 gttctactta cgtgataact tattatatat atattttctt gttatagatc cactagttct
      901 agagggacag ccccccccca aagcccccag ggatgtaatt acgtccctcc cccgctaggg
      961 ggcagcagcg agccgcccgg ggctccgctc cggtccggcg ctccccccgc atccccgagc
     1021 cggcagcgtg cggggacagc ccgggcacgg ggaaggtggc acgggatcgc tttcctctga
     1081 acgcttctcg ctgctctttg agcctgcaga cacctggggg gatacgggga aaaggcctcc
     1141 agggccgccc ccttcaccga gggcctattt cccatgattc cttcatattt gcatatacga
     1201 tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta
     1261 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1321 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     1381 tatatcttgt ggaaaggacg aaacaccggg agacgctcga cccgtctcgt ttttgcctcc
     1441 aaggcctgca ggagtcaatg ggaaaaaccc attggagcca agtacactga ctcaataggg
     1501 actttccatt gggttttgcc cagtacataa ggtcaatagg gggtgagtca acaggaaagt
     1561 cccattggag ccaagtacat tgagtcaata gggactttcc aatgggtttt gcccagtaca
     1621 taaggtcaat gggaggtaag ccaatgggtt tttcccatta ctgacatgta tactgagtca
     1681 ttagggactt tccaatgggt tttgcccagt acataaggtc aataggggtg aatcaacagg
     1741 aaagtcccat tggagccaag tacactgagt caatagggac tttccattgg gttttgccca
     1801 gtacaaaagg tcaatagggg gtgagtcaat gggtttttcc cattattggc acatacataa
     1861 ggtcaatagg ggtgactagt ggagaagagc atgcttgagg gctgagtgcc cctcagtggg
     1921 cagagagcac atggcccaca gtccctgaga agttgggggg aggggtgggc aattgaactg
     1981 gtgcctagag aaggtggggc ttgggtaaac tgggaaagtg atgtggtgta ctggctccac
     2041 ctttttcccc agggtggggg agaaccatat ataagtgcag tagtctctgt gaacattcgg
     2101 tttagtgaac ctgcaggtcg gccgccacga ccggtgccac catgactgaa tacaaaccaa
     2161 ctgttcgcct ggcaactcgt gatgatgttc cacgtgcagt tcgcaccctg gctgctgcat
     2221 ttgctgacta ccctgcaacc cgtcacactg tggacccaga ccgccacatt gaacgtgtga
     2281 ctgaactgca ggagctgttc ctgacccgtg tgggcctgga cattggcaaa gtgtgggtgg
     2341 cagatgatgg tgctgctgtg gcagtgtgga ccacccctga atctgttgaa gctggtgcag
     2401 tgtttgctga gattggccca cgcatggcag aactgtctgg cagccgcctg gcagcacaac
     2461 agcagatgga aggtctgctg gcaccacacc gcccaaaaga acctgcttgg ttcctggcaa
     2521 ctgtgggtgt gagccctgac caccagggta agggcctggg ctctgcagtg gtgctgcctg
     2581 gtgtggaagc agctgaacgt gcaggtgtgc ctgctttcct ggagacctca gctccacgca
     2641 acctgccttt ctatgaacgc ctgggcttca ctgtgactgc tgatgtggaa gtgccagaag
     2701 gcccacgcac ttggtgcatg actcgcaaac caggtgctag ccttggctcc ggcgagggca
     2761 ggggaagtct tctaacatgc ggggacgtgg aggaaaatcc cggcccactc ggctacccct
     2821 acgacgtgcc cgactacgcc agcggcatgg ccagaaaggt ggatctgaca agctgtgaca
     2881 gagaacccat ccacattcct ggcagcatcc aaccctgtgg ctgcctcctg gcttgtgatg
     2941 cacaagcagt caggattaca aggattacag aaaatgctgg agccttcttt ggaagggaaa
     3001 cccctagggt gggagagctg ttggccgatt actttggcga aacagaggcc catgccttga
     3061 gaaacgcact ggctcagagc tctgatccta aaaggcctgc tctgatcttt ggctggagag
     3121 atggcctcac tggcagaacc ttcgatatta gcctccatag gcacgatgga acctccatta
     3181 ttgaatttga gcccgctgcc gctgagcagg ctgataaccc tctgagactc acaagacaaa
     3241 tcattgccag gaccaaggaa ctcaagtccc tggaagaaat ggcagccagg gtgccaagat
     3301 atctccaggc tatgctgggc taccacagag tcatgctgta taggtttgct gatgatggaa
     3361 gcgggaaggt cattggggag gcaaagaggt ctgacctcga aagcttcttg ggacaacact
     3421 tccctgccag cctggtcccc cagcaggcca ggctgctcta tctcaagaat gccatcaggg
     3481 tggtgtctga tagcagaggg atcagctcca gaatcgtccc tgagcatgac gcctctggcg
     3541 ctgccctgga tttgagcttt gctcacctca ggagcatctc tcctatacat ttggaatttc
     3601 tgaggaacat gggggtgtct gcctccatga gcctgagcat cattatcgat ggcaccctct
     3661 ggggcctcat tatctgccac cactatgaac ctagagctgt ccctatggca cagagagtgg
     3721 ctgctgagat gttcgctgat tttctcagct tgcactttac tgctgctcat caccaaagat
     3781 aagagttcgc gtctggaaca atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3841 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3901 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3961 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     4021 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     4081 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     4141 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac
     4201 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg
     4261 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4321 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4381 ctccccgcct ggaattaatt ctggctaagc acttcgtggc cgtcgatcgt ttaaagggag
     4441 gtagtgagtc gagatatcga tactagtgat ccagacatga taagatacat tgatgagttt
     4501 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4561 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4621 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     4681 tacaaatgtg gtatggctga ttatgatcct ctagagggac agcccccccc caaagccccc
     4741 agggatgtaa ttacgtccct cccccgctag ggggcagcag cgagccgccc ggggctccgc
     4801 tccggtccgg cgctcccccc gcatccccga gccggcagcg tgcggggaca gcccgggcac
     4861 ggggaaggtg gcacgggatc gctttcctct gaacgcttct cgctgctctt tgagcctgca
     4921 gacacctggg gggatacggg gaaaaagcca taccaatgga gctcacgggg acagcccccc
     4981 cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc agcgagccgc
     5041 ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga
     5101 cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt ctcgctgctc
     5161 tttgagcctg cagacacctg gggggatacg gggaaaaaga attcgataaa agttttgtta
     5221 ctttatagaa gaaattttga gtttttgttt tttttaataa ataaataaac ataaataaat
     5281 tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc
     5341 aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa ttttacacat
     5401 gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct agctgcgtgt
     5461 tctgcagcgt gtcgagcatc ttcatctgct ccatcacgct gtaaaacaca tttgcaccgc
     5521 gagtctgccc gtcctccacg ggttcaaaaa cgtgaatgaa cgaggcgcgc tcactggccg
     5581 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5641 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5701 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg
     5761 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc
     5821 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     5881 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     5941 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     6001 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     6061 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt
     6121 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta
     6181 caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6241 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6301 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6361 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6421 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6481 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6541 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6601 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6661 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6721 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6781 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6841 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6901 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6961 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7021 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7081 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7141 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7201 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7261 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7321 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7381 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7441 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7501 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7561 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7621 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7681 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7741 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7801 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7861 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7921 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7981 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     8041 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     8101 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     8161 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     8221 aattaatgtg agttagctca ctcat
//
