LOCUS       sequence-256335-        8149 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 8149)
  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 8149)
  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..8149
                     /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"
     misc_RNA        849..924
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             2256..2309
                     /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             2316..2342
                     /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             2316..2342
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(2349..2351,2352..2354,2355..3065)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2394..2415)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2655..2674)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3002..3023
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3069..3089
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    3126..3714
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3179..3199)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3597..3608)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3794..3928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3818..3837)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3872..3891
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4706..4768
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4898..4915)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4898..4914)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4907..4929)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5056..5511
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5143..5162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5353..5374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5537..5641
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5642..5710,5711..6502)
                     /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(5860..5879)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6673..7261
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7162..7181
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7415..7432
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7549..7570
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7585..7590,7591..7608,7609..7615)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7623..7639
                     /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     7628..7650
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7647..7663
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7647..7663
                     /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 agacgctgac
      841 ccgtctctgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
      901 gaaaaagtgg caccgagtcg gtgctttttt gtttttgcct ccaaggcctg caggagtcaa
      961 tgggaaaaac ccattggagc caagtacact gactcaatag ggactttcca ttgggttttg
     1021 cccagtacat aaggtcaata gggggtgagt caacaggaaa gtcccattgg agccaagtac
     1081 attgagtcaa tagggacttt ccaatgggtt ttgcccagta cataaggtca atgggaggta
     1141 agccaatggg tttttcccat tactgacatg tatactgagt cattagggac tttccaatgg
     1201 gttttgccca gtacataagg tcaatagggg tgaatcaaca ggaaagtccc attggagcca
     1261 agtacactga gtcaataggg actttccatt gggttttgcc cagtacaaaa ggtcaatagg
     1321 gggtgagtca atgggttttt cccattattg gcacatacat aaggtcaata ggggtgacta
     1381 gtggagaaga gcatgcttga gggctgagtg cccctcagtg ggcagagagc acatggccca
     1441 cagtccctga gaagttgggg ggaggggtgg gcaattgaac tggtgcctag agaaggtggg
     1501 gcttgggtaa actgggaaag tgatgtggtg tactggctcc acctttttcc ccagggtggg
     1561 ggagaaccat atataagtgc agtagtctct gtgaacattc ggtttagtga acctgcaggt
     1621 cggccgccac gaccggtgcc accatgactg aatacaaacc aactgttcgc ctggcaactc
     1681 gtgatgatgt tccacgtgca gttcgcaccc tggctgctgc atttgctgac taccctgcaa
     1741 cccgtcacac tgtggaccca gaccgccaca ttgaacgtgt gactgaactg caggagctgt
     1801 tcctgacccg tgtgggcctg gacattggca aagtgtgggt ggcagatgat ggtgctgctg
     1861 tggcagtgtg gaccacccct gaatctgttg aagctggtgc agtgtttgct gagattggcc
     1921 cacgcatggc agaactgtct ggcagccgcc tggcagcaca acagcagatg gaaggtctgc
     1981 tggcaccaca ccgcccaaaa gaacctgctt ggttcctggc aactgtgggt gtgagccctg
     2041 accaccaggg taagggcctg ggctctgcag tggtgctgcc tggtgtggaa gcagctgaac
     2101 gtgcaggtgt gcctgctttc ctggagacct cagctccacg caacctgcct ttctatgaac
     2161 gcctgggctt cactgtgact gctgatgtgg aagtgccaga aggcccacgc acttggtgca
     2221 tgactcgcaa accaggtgct agccttggct ccggcgaggg caggggaagt cttctaacat
     2281 gcggggacgt ggaggaaaat cccggcccac tcggctaccc ctacgacgtg cccgactacg
     2341 ccagcggcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2401 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2461 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2521 ggcccaccct cgtgaccacc ttcacctacg gcgtgcagtg cttcgcccgc taccccgacc
     2581 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2641 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2701 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2761 tggggcacaa gctggagtac aactacaaca gccacaaggt ctatatcacc gccgacaagc
     2821 agaagaacgg catcaaggtg aacttcaaga cccgccacaa catcgaggac ggcagcgtgc
     2881 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     2941 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
     3001 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3061 acaagggccc caagaagaag cgcaaggtgg gcgcctaagt acaagtaaga gttcgcgtct
     3121 ggaacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     3181 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     3241 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     3301 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     3361 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     3421 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     3481 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     3541 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3601 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3661 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggaa
     3721 ttaattctgg ctaagcactt cgtggccgtc gatcgtttaa agggaggtag tgagtcgaga
     3781 tatcgatact agtgatccag acatgataag atacattgat gagtttggac aaaccacaac
     3841 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3901 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3961 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     4021 ggctgattat gatcctctag agggacagcc cccccccaaa gcccccaggg atgtaattac
     4081 gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg gtccggcgct
     4141 ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg aaggtggcac
     4201 gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca cctgggggga
     4261 tacggggaaa agccatacca atggagctca cggggacagc ccccccccaa agcccccagg
     4321 gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg gctccgctcc
     4381 ggtccggcgc tccccccgca tccccgagcc ggcagcgtgc ggggacagcc cgggcacggg
     4441 gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga gcctgcagac
     4501 acctgggggg atacggggaa aaagaattcg ataaaagttt tgttacttta tagaagaaat
     4561 tttgagtttt tgtttttttt aataaataaa taaacataaa taaattgttt gttgaattta
     4621 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa
     4681 cctcgatata cagaccgata aaacacatgc gtcaatttta cacatgatta tctttaacgt
     4741 acgtcacaat atgattatct ttctagggtt aatctagctg cgtgttctgc agcgtgtcga
     4801 gcatcttcat ctgctccatc acgctgtaaa acacatttgc accgcgagtc tgcccgtcct
     4861 ccacgggttc aaaaacgtga atgaacgagg cgcgctcact ggccgtcgtt ttacaacgtc
     4921 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     4981 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5041 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5101 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5161 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5221 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5281 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5341 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5401 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5461 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     5521 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5581 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5641 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5701 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5761 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5821 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5881 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5941 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6001 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6061 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6121 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6181 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6241 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6301 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6361 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6421 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6481 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6541 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6601 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6661 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6721 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6781 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6841 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6901 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6961 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7021 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7081 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7141 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7201 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7261 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7321 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     7381 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     7441 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     7501 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7561 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7621 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     7681 cgcccgccgg gtaactcacg gggtatccat gtccatttct gcggcatcca gccaggatac
     7741 ccgtcctcgc tgacgtaata tcccagcgcc gcaccgctgt cattaatctg cacaccggca
     7801 cggcagttcc ggctgtcgcc ggtattgttc gggttgctga tgcgcttcgg gctgaccatc
     7861 cggaactgtg tccggaaaag ccgcgacgaa ctggtatccc aggtggcctg aacgaacagt
     7921 tcaccgttaa aggcgtgcat ggccacacct tcccgaatca tcatggtaaa cgtgcgtttt
     7981 cgctcaacgt caatgcagca gcagtcatcc tcggcaaact ctttccatgc cgcttcaacc
     8041 tcgcgggaaa aggcacgggc ttcttcctcc ccgatgccca gatagcgcca gcttgggcga
     8101 tgactgagcc ggaaaaaaga cccgacgata tgatcctgat gcagctaga
//
