LOCUS       sequence-200489-        7779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector for sgRNA cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7779)
  AUTHORS   Ishida K, Xu H, Sasakawa N, Lung MSY, Kudryashev JA, Gee P, Hotta A
  TITLE     Site-specific randomization of the endogenous genome by a 
            regulatable CRISPR-Cas9 piggyBac system in human cells.
  JOURNAL   Sci Rep. 2018 Jan 10;8(1):310. doi: 10.1038/s41598-017-18568-4.
  PUBMED    29321585
REFERENCE   2  (bases 1 to 7779)
  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: "Sci Rep."; 
            date: "2018-01-10"; volume: "8(1):310. doi"; pages: " 
            10.1038/s41598-017-18568-4"
COMMENT     Sequence Label: pPV-H1-ccdB-mEF1-alpha-RiH
FEATURES             Location/Qualifiers
     source          1..7779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   46..108
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        452..666
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     587..606
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     CDS             1004..1309
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1188..1207
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1350..1474)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     intron          1709..2656
                     /label=mEF-1-alpha intron
                     /note="intron upstream of the start codon of mouse 
                     EF-1-alpha"
     CDS             2708..3385
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2708..3385
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2838..2856)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3106..3125
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3300..3323
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    3394..3838
                     /label=FMDV IRES
                     /note="internal ribosome entry site (IRES) of the 
                     foot-and-mouth disease virus"
     promoter        3872..3918
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        3910..3928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3938..4963
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     repeat_region   5746..5780
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5873..5890)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6044..6632)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6124..6143)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6803..7594,7595..7663))
                     /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     7426..7445
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7664..7768)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc atctgttaac attatacgcg tttaacccta gaaagataat
       61 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      121 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      181 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      241 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      301 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
      361 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
      421 cacatatcaa tattatgctc tgctagcgtt tgaacgctga cgtcatcaac ccgctccaag
      481 gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa
      541 gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct
      601 gggaaatcac cataaacgtg aaatgtcttt ggatttggga atcttataag ttctgtatga
      661 gaccacttgg atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt
      721 tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc
      781 tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata
      841 tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg
      901 cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt
      961 gaaatgaacg gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta
     1021 cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga
     1081 cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt
     1141 ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac
     1201 cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg
     1261 cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc
     1321 ccttatacac agccagtctg caggtcgacc atagtgactg gatatgttgt gttttacagt
     1381 attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt
     1441 acgtttctcg ttcagctttc ttgtacaaag tggtgatcta gctaaaacgc ggcctcgaat
     1501 cgaattcaaa cccgggcgat cgctctagac tagtgcggcc gcccctcccc cacagcaggg
     1561 gcggggtccc gcgcccaccg gaaggagcgg gctcggggcg ggcggcgctg attggccggg
     1621 gcgggcctga cgccgacgcg gctataagag accacaagcg acccgcaggg ccagacgttc
     1681 ttcgccgaag cttgccgtca gaacgcaggt gaggggcggg tgtggcttcc gcgggccgcc
     1741 gagctggagg tcctgctccg agcgggccgg gccccgctgt cgtcggcggg gattagctgc
     1801 gagcattccc gcttcgagtt gcgggcggcg cgggaggcag agtgcgaggc ctagcggcaa
     1861 ccccgtagcc tcgcctcgtg tccggcttga ggcctagcgt ggtgtccgcg ccgccgccgc
     1921 gtgctactcc ggccgcactc tggtcttttt tttttttgtt gttgttgccc tgctgccttc
     1981 gattgccgtt cagcaatagg ggctaacaaa gggagggtgc ggggcttgct cgcccggagc
     2041 ccggagaggt catggttggg gaggaatgga gggacaggag tggcggctgg ggcccgcccg
     2101 ccttcggagc acatgtccga cgccacctgg atggggcgag gcctggggtt tttcccgaag
     2161 caaccaggct ggggttagcg tgccgaggcc atgtggcccc agcacccggc acgatctggc
     2221 ttggcggcgc cgcgttgccc tgcctcccta actagggtga ggccatcccg tccggcacca
     2281 gttgcgtgcg tggaaagatg gccgctcccg ggccctgttg caaggagctc aaaatggagg
     2341 acgcggcagc ccggtggagc gggcgggtga gtcacccaca caaaggaaga gggcctggtc
     2401 cctcaccggc tgctgcttcc tgtgaccccg tggtcctatc ggccgcaata gtcacctcgg
     2461 gcttttgagc acggctagtc gcggcggggg gaggggatgt aatggcgttg gagtttgttc
     2521 acatttggtg ggtggagact agtcaggcca gcctggcgct ggaagtcatt tttggaattt
     2581 gtccccttga gttttgagcg gagctaattc tcgggcttct tagcggttca aaggtatctt
     2641 ttaaaccctt ttttaggtgt tgtgaaaacc accgctaatt caaagcaacc ggtgatatcg
     2701 gatcaccatg gcctcctccg aggacgtcat caaggagttc atgcgcttca aggtgcgcat
     2761 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2821 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     2881 ggacatcctg tcccctcagt tccagtacgg ctccaaggcc tacgtgaagc accccgccga
     2941 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     3001 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     3061 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     3121 gaagaccatg ggctgggagg cctccaccga gcggatgtac cccgaggacg gcgccctgaa
     3181 gggcgagatc aagatgaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa
     3241 gaccacctac atggccaaga agcccgtgca gctgcccggc gcctacaaga ccgacatcaa
     3301 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga
     3361 gggccgccac tccaccggcg cctaatacct aggagcaggt ttccccaatg acacaaaacg
     3421 tgcaacttga aactccgcct ggtctttcca ggtctagagg ggtaacactt tgtactgcgt
     3481 ttggctccac gctcgatcca ctggcgagtg ttagtaacag cactgttgct tcgtagcgga
     3541 gcatgacggc cgtgggaact cctccttggt aacaaggacc cacggggcca aaagccacgc
     3601 ccacacgggc ccgtcatgtg tgcaacccca gcacggcgac tttactgcga aacccacttt
     3661 aaagtgacat tgaaactggt acccacacac tggtgacagg ctaaggatgc ccttcaggta
     3721 ccccgaggta acacgcgaca ctcgggatct gagaagggga ctggggcttc tataaaagcg
     3781 ctcggtttaa aaagcttcta tgcctgaata ggtgaccgga ggtcggcacc tttcctttgc
     3841 aattactgac cctatgaata caactgactg tttgacaatt aatcatcggc atagtatatc
     3901 ggcatagtat aatacgactc actataggag ggccaccatg aagaaacctg aactgacagc
     3961 aacttctgtt gagaagtttc tcattgaaaa atttgattct gtttctgatc tcatgcagct
     4021 gtctgaaggt gaagaaagca gagccttttc ttttgatgtt ggaggaagag gttatgttct
     4081 gagggtcaat tcttgtgctg atggttttta caaagacaga tatgtttaca gacactttgc
     4141 ctctgctgct ctgccaattc cagaagttct ggacattgga gaattttctg aatctctcac
     4201 ctactgcatc agcagaagag cacaaggagt cactctccag gatctccctg aaactgagct
     4261 gccagctgtt ctgcaacctg ttgctgaagc aatggatgcc attgcagcag ctgatctgag
     4321 ccaaacctct ggatttggtc cttttggtcc ccaaggcatt ggtcagtaca ccacttggag
     4381 ggatttcatt tgtgccattg ctgatcctca tgtctatcac tggcagactg tgatggatga
     4441 cacagtttct gcttctgttg ctcaggcact ggatgaactc atgctgtggg cagaagattg
     4501 tcctgaagtc agacacctgg tccatgctga ttttggaagc aacaatgttc tgacagacaa
     4561 tggcagaatc actgcagtca ttgactggtc tgaagccatg tttggagatt ctcaatatga
     4621 ggttgccaac attttttttt ggagaccttg gctggcttgc atggaacaac aaacaagata
     4681 ttttgaaaga agacacccag aactggctgg ttcccccaga ctgagagcct acatgctcag
     4741 aattggcctg gaccaactgt atcaatctct ggttgatgga aactttgatg atgctgcttg
     4801 ggcacaagga agatgtgatg ccattgtgag gtctggtgct ggaactgttg gaagaactca
     4861 aattgcaaga aggtctgctg ctgtttggac tgatggatgt gttgaagttc tggctgactc
     4921 tggaaacagg agaccctcca caagacccag agccaaggaa tgaatattag ctagattatc
     4981 cctaatacct gccaccccac tcttaatcag tggtggaaga acggtctcag aactgtttgt
     5041 ttcaattggc catttaagtt tagtagtaaa agactggtta atgataacaa tgcatcgtaa
     5101 aaccttcaga aggaaaggag aatgttttgt ggaccacttt ggttttcttt tttgcgtgtg
     5161 gcagttttaa gttattagtt tttaaaatca gtacttttta atggaaacaa cttgaccaaa
     5221 aatttgtcac agaattttga gacccattaa aaaagttaaa tgagaaacct gtgtgttcct
     5281 ttggtcaaca ccgagacatt taggtgaaag acatctaatt ctggttttac gaatctggaa
     5341 acttcttgaa aatgtaattc ttgagtttac ctcgagttaa ttaaccaaca agctcgtcat
     5401 cgctttgcag aagagcagag aggatatgct catcgtctaa agaactaccc attttattat
     5461 atattagtca cgatatctat aacaagaaaa tatatatata ataagttatc acgtaagtag
     5521 aacatgaaat aacaatataa ttatcgtatg agttaaatct taaaagtcac gtaaaagata
     5581 atcatgcgtc attttgactc acgcggtcgt tatagttcaa aatcagtgac acttaccgca
     5641 ttgacaagca cgcctcacgg gagctccaag cggcgactga gatgtcctaa atgcacagcg
     5701 acggattcgc gctatttaga aagagagagc aatatttcaa gaatgcatgc gtcaatttta
     5761 cgcagactat ctttctaggg ttaatacgta taatacatat gattcagctg cattaatgaa
     5821 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     5881 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     5941 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     6001 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6061 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6121 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6181 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6241 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6301 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6361 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6421 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6481 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6541 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6601 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6661 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     6721 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     6781 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     6841 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6901 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6961 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7021 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7081 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7141 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7201 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7261 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7321 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7381 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7441 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7501 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7561 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7621 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7681 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     7741 agaaaaataa acaaataggg gttccgcgca catttcccc
//
