LOCUS       sequence-408798-        7130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector for crRNA cloning and expression. Blasticidin 
            selectable..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7130)
  AUTHORS   Kita Y, Okuzaki Y, Naoe Y, Lee J, Bang U, Okawa N, Ichiki A, 
            Jonouchi T, Sakurai H, Kojima Y, Hotta A
  TITLE     Dual CRISPR-Cas3 system for inducing multi-exon skipping in DMD 
            patient-derived iPSCs.
  JOURNAL   Stem Cell Reports. 2023 Sep 12;18(9):1753-1765. doi: 
            10.1016/j.stemcr.2023.07.007. Epub 2023 Aug 24.
  PUBMED    37625413
REFERENCE   2  (bases 1 to 7130)
  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"; doi: 
            "10.1016/j.stemcr.2023.07.007"; journalName: "Stem Cell Reports"; 
            date: "2023-09-12- 12"; volume: "18"; issue: "9"; pages: "1753-1765"
COMMENT     Sequence Label: pPV-C1-crRNA-cloning-EF1a-BA
FEATURES             Location/Qualifiers
     source          1..7130
                     /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)"
     primer_bind     468..484
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     468..484
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(612..917)
                     /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     complement(714..733)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1259..1918)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1833..1852
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1972..1978,1979..1996,1997..2002))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(2214..2233)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2503..3681
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2734..3672
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3629..3649
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3704..3728
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3757..3766
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3763..4161
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISQDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4274..4293)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4339..4394
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4393..4412)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     repeat_region   5097..5131
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5224..5241)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5395..5983)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5475..5494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6154..6945,6946..7014))
                     /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     6777..6796
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7015..7119)
                     /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 taaacccggg cgatcgctct agaactagtg
      481 gatcaatgat acggcgacca ccgagatctc cccgggctgc aggaattcaa aaaacggttt
      541 atccccgctg gcgcggggaa cacaggtctt cgctagcctg cagactggct gtgtataagg
      601 gagcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat gtcattttcg
      661 cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact ggccatatcg
      721 gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag ttcacgggag
      781 actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg cccgggcgtg
      841 tcaataatat cactctgtac atccacaaac agacgataac ggctctctct tttataggtg
      901 taaaccttaa actgcatttc accagcccct gttctcgtca gcaaaagagc cgttcatttc
      961 aataaaccgg gcgacctcag ccatcccttc ctgattttcc gctttccagc gttcggcacg
     1021 cagacgacgg gcttcattct gcatggttgt gcttaccaga ccggagatat tgacatcata
     1081 tatgccttga gcaactgata gctgtcgctg tcaactgtca ctgtaatacg ctgcttcata
     1141 gcatacctct ttttgacata cttcgggtat acatatcagt atatattctt ataccgcaaa
     1201 aatcagcgcg caaatacgca tactgttatc tggcttttag taagccggat ccacgcgttt
     1261 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     1321 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     1381 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     1441 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     1501 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     1561 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     1621 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     1681 cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa
     1741 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     1801 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     1861 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt
     1921 tagcttcctt agctcctgaa aatctcgacg gatcctaact caaaatccac acattatacg
     1981 agccggaagc ataaagtgta aagcctgggg tgcctaatgc ggccgcctag cgaagacccc
     2041 ggtttatccc cgctggcgcg gggaacactc taaacataac ctattattaa ttaatgattt
     2101 tttaagccag tcacaatcta ccaactttat agtatcacac aaacaacaca tccacggtgt
     2161 ttcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     2221 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     2281 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattccaa
     2341 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaagaattc
     2401 acgtcaattc gatatcaagc tttgcaaaga tggataaagt tttaaacaga gaggaatctt
     2461 tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg tgcccgtcag
     2521 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     2581 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     2641 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     2701 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     2761 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag
     2821 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc
     2881 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc
     2941 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc
     3001 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat
     3061 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc
     3121 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc
     3181 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt
     3241 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga
     3301 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag
     3361 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca
     3421 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg
     3481 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag
     3541 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc
     3601 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt
     3661 cttccatttc aggtgtcgtg aggaattagc ggtggcggcc atcacaagtt tgtacaaaaa
     3721 agcaggctgg cgccggaacc aattcaggaa ttcgccgcca ccatggccaa gcctttgtct
     3781 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
     3841 caagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
     3901 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
     3961 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
     4021 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
     4081 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
     4141 ggttatgtgt gggagggcta aggatcctag gcttaagtat acctcgagtt aaattcactc
     4201 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     4261 aataccactg agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg
     4321 agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt
     4381 ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg
     4441 agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt
     4501 ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata
     4561 gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt
     4621 taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac
     4681 tactcccagt catagctgtc cctcttctct tatggagatc cctcgactta attaaccaac
     4741 aagctcgtca tcgctttgca gaagagcaga gaggatatgc tcatcgtcta aagaactacc
     4801 cattttatta tatattagtc acgatatcta taacaagaaa atatatatat aataagttat
     4861 cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca
     4921 cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga
     4981 cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta
     5041 aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg
     5101 cgtcaatttt acgcagacta tctttctagg gttaatacgt ataatacata tgattcagct
     5161 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5221 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5281 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5341 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5401 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5461 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5521 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5581 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5641 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5701 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5761 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5821 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5881 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5941 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6001 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6061 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6121 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6181 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6241 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6301 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6361 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6421 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6481 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6541 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6601 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6661 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6721 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6781 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6841 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6901 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6961 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7021 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7081 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
//
