LOCUS       sequence-267392-        8150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Piggybac vector for CRISPR/SpCas9 applications (nuclease, base 
            editing, or epigenetic modifications), co-expressed with GFP-NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8150)
  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 8150)
  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-gR-PnGW
FEATURES             Location/Qualifiers
     source          1..8150
                     /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"
     misc_RNA        1414..1489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2821..2874
                     /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             2881..2907
                     /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             2881..2907
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2914..2916,2917..2919,2920..3630)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2959..2980)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3220..3239)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3567..3588
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3634..3654
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3691..4279
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3744..3764)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4162..4173)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4359..4493
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4383..4402)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4437..4456
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5272..5334
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5464..5481)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5464..5480)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5473..5495)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5622..6077
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5709..5728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5919..5940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6103..6207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6208..6276,6277..7068)
                     /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(6426..6445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7239..7827
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7728..7747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7981..7998
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8115..8136
                     /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 accggagacg ctgacccgtc tctgttttag agctagaaat agcaagttaa
     1441 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt
     1501 tgcctccaag gcctgcagga gtcaatggga aaaacccatt ggagccaagt acactgactc
     1561 aatagggact ttccattggg ttttgcccag tacataaggt caataggggg tgagtcaaca
     1621 ggaaagtccc attggagcca agtacattga gtcaataggg actttccaat gggttttgcc
     1681 cagtacataa ggtcaatggg aggtaagcca atgggttttt cccattactg acatgtatac
     1741 tgagtcatta gggactttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat
     1801 caacaggaaa gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt
     1861 ttgcccagta caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcaca
     1921 tacataaggt caataggggt gactagtgga gaagagcatg cttgagggct gagtgcccct
     1981 cagtgggcag agagcacatg gcccacagtc cctgagaagt tggggggagg ggtgggcaat
     2041 tgaactggtg cctagagaag gtggggcttg ggtaaactgg gaaagtgatg tggtgtactg
     2101 gctccacctt tttccccagg gtgggggaga accatatata agtgcagtag tctctgtgaa
     2161 cattcggttt agtgaacctg caggtcggcc gccacgaccg gtgccaccat gactgaatac
     2221 aaaccaactg ttcgcctggc aactcgtgat gatgttccac gtgcagttcg caccctggct
     2281 gctgcatttg ctgactaccc tgcaacccgt cacactgtgg acccagaccg ccacattgaa
     2341 cgtgtgactg aactgcagga gctgttcctg acccgtgtgg gcctggacat tggcaaagtg
     2401 tgggtggcag atgatggtgc tgctgtggca gtgtggacca cccctgaatc tgttgaagct
     2461 ggtgcagtgt ttgctgagat tggcccacgc atggcagaac tgtctggcag ccgcctggca
     2521 gcacaacagc agatggaagg tctgctggca ccacaccgcc caaaagaacc tgcttggttc
     2581 ctggcaactg tgggtgtgag ccctgaccac cagggtaagg gcctgggctc tgcagtggtg
     2641 ctgcctggtg tggaagcagc tgaacgtgca ggtgtgcctg ctttcctgga gacctcagct
     2701 ccacgcaacc tgcctttcta tgaacgcctg ggcttcactg tgactgctga tgtggaagtg
     2761 ccagaaggcc cacgcacttg gtgcatgact cgcaaaccag gtgctagcct tggctccggc
     2821 gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccactcggc
     2881 tacccctacg acgtgcccga ctacgccagc ggcatggtga gcaagggcga ggagctgttc
     2941 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3001 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3061 accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcac ctacggcgtg
     3121 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3181 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3241 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3301 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3361 aaggtctata tcaccgccga caagcagaag aacggcatca aggtgaactt caagacccgc
     3421 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3481 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3541 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3601 atcactctcg gcatggacga gctgtacaag ggccccaaga agaagcgcaa ggtgggcgcc
     3661 taagtacaag taagagttcg cgtctggaac aatcaacctc tggattacaa aatttgtgaa
     3721 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3781 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3841 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3901 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3961 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4021 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     4081 gggaagctga cgtcctttcc atggctgctc gcctgtgttg ccacctggat tctgcgcggg
     4141 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4201 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4261 ctttgggccg cctccccgcc tggaattaat tctggctaag cacttcgtgg ccgtcgatcg
     4321 tttaaaggga ggtagtgagt cgagatatcg atactagtga tccagacatg ataagataca
     4381 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     4441 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     4501 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     4561 agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctagaggga cagccccccc
     4621 ccaaagcccc cagggatgta attacgtccc tcccccgcta gggggcagca gcgagccgcc
     4681 cggggctccg ctccggtccg gcgctccccc cgcatccccg agccggcagc gtgcggggac
     4741 agcccgggca cggggaaggt ggcacgggat cgctttcctc tgaacgcttc tcgctgctct
     4801 ttgagcctgc agacacctgg ggggatacgg ggaaaaagcc ataccaatgg agctcacggg
     4861 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag
     4921 cagcgagccg cccggggctc cgctccggtc cggcgctccc cccgcatccc cgagccggca
     4981 gcgtgcgggg acagcccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct
     5041 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag aattcgataa
     5101 aagttttgtt actttataga agaaattttg agtttttgtt ttttttaata aataaataaa
     5161 cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt
     5221 aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca
     5281 attttacaca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc
     5341 tagctgcgtg ttctgcagcg tgtcgagcat cttcatctgc tccatcacgc tgtaaaacac
     5401 atttgcaccg cgagtctgcc cgtcctccac gggttcaaaa acgtgaatga acgaggcgcg
     5461 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     5521 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     5581 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     5641 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5701 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5761 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5821 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5881 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5941 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     6001 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     6061 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     6121 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6181 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6241 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6301 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6361 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6421 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6481 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6541 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6601 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6661 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6721 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6781 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6841 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6901 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6961 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     7021 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     7081 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7141 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7201 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7261 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7321 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7381 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7441 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7501 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7561 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7621 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7681 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7741 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7801 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7861 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7921 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7981 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     8041 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     8101 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
//
