LOCUS       sequence-97111-e        6946 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for CRISPR-sgRNA (into the BamHI-EcoRI site), 
            expresses RFP and hygromycin resistance gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6946)
  AUTHORS   Li HL, Fujimoto N, Sasakawa N, Shirai S, Ohkame T, Sakuma T, Tanaka 
            M, Amano N, Watanabe A, Sakurai H, Yamamoto T, Yamanaka S, Hotta A
  TITLE     Precise Correction of the Dystrophin Gene in Duchenne Muscular 
            Dystrophy Patient Induced Pluripotent Stem Cells by TALEN and 
            CRISPR-Cas9.
  JOURNAL   Stem Cell Reports. 2014 Nov 25. pii: S2213-6711(14)00335-X. doi: 
            10.1016/j.stemcr.2014.10.013.
  PUBMED    25434822
REFERENCE   2  (bases 1 to 6946)
  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: "Stem Cell 
            Reports. 2014 Nov 25. pii: S2213-6711(14)00335-X. doi: 
            10.1016/j.stemcr.2014.10.013."
COMMENT     Sequence Label: pHL-H1-ccdB-mEF1a-RiH
FEATURES             Location/Qualifiers
     source          1..6946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        31..245
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     166..185
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     CDS             583..888
                     /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     767..786
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(929..1053)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     intron          1288..2235
                     /label=mEF-1-alpha intron
                     /note="intron upstream of the start codon of mouse 
                     EF-1-alpha"
     CDS             2287..2964
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2287..2964
                     /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(2417..2435)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2685..2704
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2879..2902
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    2973..3417
                     /label=FMDV IRES
                     /note="internal ribosome entry site (IRES) of the 
                     foot-and-mouth disease virus"
     promoter        3451..3497
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        3489..3507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3517..4542
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5040..5057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5211..5799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5291..5310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5970..6761,6762..6830))
                     /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     6593..6612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6831..6935)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gctagcgttt gaacgctgac gtcatcaacc cgctccaagg
       61 aatcgcgggc ccagtgtcac taggcgggaa cacccagcgc gcgtgcgccc tggcaggaag
      121 atggctgtga gggacagggg agtggcgccc tgcaatattt gcatgtcgct atgtgttctg
      181 ggaaatcacc ataaacgtga aatgtctttg gatttgggaa tcttataagt tctgtatgag
      241 accacttgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
      301 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
      361 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
      421 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
      481 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
      541 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
      601 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
      661 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
      721 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
      781 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
      841 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
      901 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
      961 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     1021 cgtttctcgt tcagctttct tgtacaaagt ggtgatctag ctaaaacgcg gcctcgaatc
     1081 gaattcaaac ccgggcgatc gctctagact agtgcggccg cccctccccc acagcagggg
     1141 cggggtcccg cgcccaccgg aaggagcggg ctcggggcgg gcggcgctga ttggccgggg
     1201 cgggcctgac gccgacgcgg ctataagaga ccacaagcga cccgcagggc cagacgttct
     1261 tcgccgaagc ttgccgtcag aacgcaggtg aggggcgggt gtggcttccg cgggccgccg
     1321 agctggaggt cctgctccga gcgggccggg ccccgctgtc gtcggcgggg attagctgcg
     1381 agcattcccg cttcgagttg cgggcggcgc gggaggcaga gtgcgaggcc tagcggcaac
     1441 cccgtagcct cgcctcgtgt ccggcttgag gcctagcgtg gtgtccgcgc cgccgccgcg
     1501 tgctactccg gccgcactct ggtctttttt ttttttgttg ttgttgccct gctgccttcg
     1561 attgccgttc agcaataggg gctaacaaag ggagggtgcg gggcttgctc gcccggagcc
     1621 cggagaggtc atggttgggg aggaatggag ggacaggagt ggcggctggg gcccgcccgc
     1681 cttcggagca catgtccgac gccacctgga tggggcgagg cctggggttt ttcccgaagc
     1741 aaccaggctg gggttagcgt gccgaggcca tgtggcccca gcacccggca cgatctggct
     1801 tggcggcgcc gcgttgccct gcctccctaa ctagggtgag gccatcccgt ccggcaccag
     1861 ttgcgtgcgt ggaaagatgg ccgctcccgg gccctgttgc aaggagctca aaatggagga
     1921 cgcggcagcc cggtggagcg ggcgggtgag tcacccacac aaaggaagag ggcctggtcc
     1981 ctcaccggct gctgcttcct gtgaccccgt ggtcctatcg gccgcaatag tcacctcggg
     2041 cttttgagca cggctagtcg cggcgggggg aggggatgta atggcgttgg agtttgttca
     2101 catttggtgg gtggagacta gtcaggccag cctggcgctg gaagtcattt ttggaatttg
     2161 tccccttgag ttttgagcgg agctaattct cgggcttctt agcggttcaa aggtatcttt
     2221 taaacccttt tttaggtgtt gtgaaaacca ccgctaattc aaagcaaccg gtgatatcgg
     2281 atcaccatgg cctcctccga ggacgtcatc aaggagttca tgcgcttcaa ggtgcgcatg
     2341 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2401 gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg
     2461 gacatcctgt cccctcagtt ccagtacggc tccaaggcct acgtgaagca ccccgccgac
     2521 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     2581 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     2641 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     2701 aagaccatgg gctgggaggc ctccaccgag cggatgtacc ccgaggacgg cgccctgaag
     2761 ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc cgaggtcaag
     2821 accacctaca tggccaagaa gcccgtgcag ctgcccggcg cctacaagac cgacatcaag
     2881 ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgcgccgag
     2941 ggccgccact ccaccggcgc ctaataccta ggagcaggtt tccccaatga cacaaaacgt
     3001 gcaacttgaa actccgcctg gtctttccag gtctagaggg gtaacacttt gtactgcgtt
     3061 tggctccacg ctcgatccac tggcgagtgt tagtaacagc actgttgctt cgtagcggag
     3121 catgacggcc gtgggaactc ctccttggta acaaggaccc acggggccaa aagccacgcc
     3181 cacacgggcc cgtcatgtgt gcaaccccag cacggcgact ttactgcgaa acccacttta
     3241 aagtgacatt gaaactggta cccacacact ggtgacaggc taaggatgcc cttcaggtac
     3301 cccgaggtaa cacgcgacac tcgggatctg agaaggggac tggggcttct ataaaagcgc
     3361 tcggtttaaa aagcttctat gcctgaatag gtgaccggag gtcggcacct ttcctttgca
     3421 attactgacc ctatgaatac aactgactgt ttgacaatta atcatcggca tagtatatcg
     3481 gcatagtata atacgactca ctataggagg gccaccatga agaaacctga actgacagca
     3541 acttctgttg agaagtttct cattgaaaaa tttgattctg tttctgatct catgcagctg
     3601 tctgaaggtg aagaaagcag agccttttct tttgatgttg gaggaagagg ttatgttctg
     3661 agggtcaatt cttgtgctga tggtttttac aaagacagat atgtttacag acactttgcc
     3721 tctgctgctc tgccaattcc agaagttctg gacattggag aattttctga atctctcacc
     3781 tactgcatca gcagaagagc acaaggagtc actctccagg atctccctga aactgagctg
     3841 ccagctgttc tgcaacctgt tgctgaagca atggatgcca ttgcagcagc tgatctgagc
     3901 caaacctctg gatttggtcc ttttggtccc caaggcattg gtcagtacac cacttggagg
     3961 gatttcattt gtgccattgc tgatcctcat gtctatcact ggcagactgt gatggatgac
     4021 acagtttctg cttctgttgc tcaggcactg gatgaactca tgctgtgggc agaagattgt
     4081 cctgaagtca gacacctggt ccatgctgat tttggaagca acaatgttct gacagacaat
     4141 ggcagaatca ctgcagtcat tgactggtct gaagccatgt ttggagattc tcaatatgag
     4201 gttgccaaca tttttttttg gagaccttgg ctggcttgca tggaacaaca aacaagatat
     4261 tttgaaagaa gacacccaga actggctggt tcccccagac tgagagccta catgctcaga
     4321 attggcctgg accaactgta tcaatctctg gttgatggaa actttgatga tgctgcttgg
     4381 gcacaaggaa gatgtgatgc cattgtgagg tctggtgctg gaactgttgg aagaactcaa
     4441 attgcaagaa ggtctgctgc tgtttggact gatggatgtg ttgaagttct ggctgactct
     4501 ggaaacagga gaccctccac aagacccaga gccaaggaat gaatattagc tagattatcc
     4561 ctaatacctg ccaccccact cttaatcagt ggtggaagaa cggtctcaga actgtttgtt
     4621 tcaattggcc atttaagttt agtagtaaaa gactggttaa tgataacaat gcatcgtaaa
     4681 accttcagaa ggaaaggaga atgttttgtg gaccactttg gttttctttt ttgcgtgtgg
     4741 cagttttaag ttattagttt ttaaaatcag tactttttaa tggaaacaac ttgaccaaaa
     4801 atttgtcaca gaattttgag acccattaaa aaagttaaat gagaaacctg tgtgttcctt
     4861 tggtcaacac cgagacattt aggtgaaaga catctaattc tggttttacg aatctggaaa
     4921 cttcttgaaa atgtaattct tgagtttacc ctcgagatta attaaaccgg tcagctgcat
     4981 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5041 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5101 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5161 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5221 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5281 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5341 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5401 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5461 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5521 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5581 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5641 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5701 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5761 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5821 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5881 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5941 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6001 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6061 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6121 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6181 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6241 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6301 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6361 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6421 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6481 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6541 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6601 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6661 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6721 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6781 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6841 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6901 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttcccc
//
