LOCUS       sequence-275228-        6256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector; Can be combined with pBFv-U6.2 to generate 
            double-gRNA expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6256)
  AUTHORS   Kondo S, Ueda R
  TITLE     Highly improved gene targeting by germline-specific Cas9 expression 
            in Drosophila.
  JOURNAL   Genetics. 2013 Nov;195(3):715-21. doi: 10.1534/genetics.113.156737. 
            Epub 2013 Sep 3.
  PUBMED    24002648
REFERENCE   2  (bases 1 to 6256)
  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: 
            "10.1534/genetics.113.156737"; journalName: "Genetics"; date: 
            "2013-11"; volume: "195"; issue: "3"; pages: "715-21"
COMMENT     Sequence Label: pBFv-U6.2B
FEATURES             Location/Qualifiers
     source          1..6256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(252..268)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(252..268)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        277..675
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        697..772
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(849..869)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(849..867)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(888..904)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(888..904)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(901..923)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    912..928
                     /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)."
     promoter        complement(join(936..942,943..960,961..966))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    981..1002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1119..1136)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1290..1878)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1370..1389)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2049..2840,2841..2909))
                     /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     2672..2691
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2910..3014)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3040..3495)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3177..3198)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3389..3408
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3622..3644
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3636..3653
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3637..3653
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3663..3682
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3663..3681
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3889..3958
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             complement(join(4158..4275,4352..4445,4537..5143,5198..5331,
                     5392..5552,5626..5651))
                     /codon_start=1
                     /product="tryptophan oxygenase"
                     /label=vermilion
                     /note="selectable marker, required to synthesize the brown 
                     eye pigment in Drosophila"
                     /translation="MSCPYAGNGNDHDDSAVPLTTEVGKIYGEYLMLDKLLDAQCMLSE
                     EDKRPVHDEHLFIITHQAYELWFKQIIFEFDSIRDMLDAEVIDETKTLEIVKRLNRVVL
                     ILKLLVDQVPILETMTPLDFMDFRKYLAPASGFQSLQFRLIENKLGVLTEQRVRYNQKY
                     SDVFSDEEARNSIRNSEKDPSLLELVQRWLERTPGLEESGFNFWAKFQESVDRFLEAQV
                     QSAMEEPVEKAKNYRLMDIEKRREVYRSIFDPAVHDALVRRGDRRFSHRALQGAIMITF
                     YRDEPRFSQPHQLLTLLMDIDSLITKWRYNHVIMVQRMIGSQQLGTGGSSGYQYLRSTL
                     SDRYKVFLDLFNLSTFLIPREAIPPLDETIRKKLINKSV"
ORIGIN
        1 attcggttac catgatactt cgacgcataa ctgtagattt tggatagaat taaagagaaa
       61 atggcgagag agtaaaattc cggcgtcggc aaagtagagc aaaaaaatca gtataccatt
      121 tagctacctc tctcactcgc acgcagtgcc ggctcaagtt gggcgcggct ctgcaattat
      181 cgattttctt ggggtgtgta actaatcatc cgttttccct tcctcctcat ccacagcgtg
      241 aaggcgcgcc ggatccacta gttctagagc ggccgcttcg acttgcagcc tgaaatacgg
      301 cacgagtagg aaaagccgag tcaaatgccg aatgcagagt ctcattacag cacaatcaac
      361 tcaagaaaaa ctcgacactt ttttaccatt tgcacttaaa tcctttttta ttcgttatgt
      421 atactttttt tggtccctaa ccaaaacaaa accaaactct cttagtcgtg cctctatatt
      481 taaaactatc aatttattat agtcaataaa tcgaactgtg ttttcaacaa acgaacaata
      541 ggacactttg attctaaagg aaattttgaa aatcttaagc agagggttct taagaccatt
      601 tgccaattct tataattctc aactgctctt tcctgatgtt gatcatttat ataggtatgt
      661 tttcctcaat acttcgggtc ttcgagttga agacctgttt tagagctaga aatagcaagt
      721 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
      781 catgcacttg gctttccacc gttggtatcg attctctggg acgatgagtc gagctccagc
      841 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
      901 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
      961 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     1021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     1081 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     1141 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     1201 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     1261 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1321 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1381 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1441 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1501 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1561 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1621 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1681 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1741 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1801 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1861 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1921 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1981 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2041 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2101 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2161 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     2221 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2281 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2341 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2401 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2461 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2521 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2581 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2641 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2701 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2761 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2821 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2881 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2941 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3001 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     3061 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3121 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3181 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3241 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3301 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3361 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3421 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3481 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     3541 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     3601 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg
     3661 cgtaatacga ctcactatag ggcgaattgg gtacctcgac atgcccgccg tgaccgtcga
     3721 gaacccgctg acgctgcccc gcgtatccgc acccgccgac gccgtcgcac gtcccgtgct
     3781 caccgtgacc accgcgccca gcggtttcga gggcgagggc ttcccggtgc gccgcgcgtt
     3841 cgccgggatc aactaccgcc acctcgaccc gttcatcatg atggaccaga tgggtgaggt
     3901 ggagtacgcg cccggggagc ccaagggcac gccctggcac ccgcaccgcg gcttcgagac
     3961 cgtgacctac atcgtcgaca agcttggatt tattttgtta tgttatatgt attatatgtc
     4021 agacataaag aaaaggaaca catcaaatgt gataacaaag actaaacaag taattttatt
     4081 acaccaaaac gacaaaacag taggcagaac aaacaacgca tagccaaaca ttgacgaatt
     4141 ggataccctg ccgattgtca gacacttttg ttgatcagtt tcttgcgaat ggtctcgtcc
     4201 agcggtggaa tcgcctcgcg gggaatcaga aaagtggaca gattgaacag atccagaaac
     4261 accttgtacc gatcactgaa accaaaaaaa aacaaaggga gaacagtttg agttcattga
     4321 tccccgatat aatcacatct gcgatgatca cctgagagtg gagcgcagat attgatatcc
     4381 agacgagcca ccagtgccca actgttggga tccaatcatg cgttgcacca tgatcacgtg
     4441 attgtctgcg gcgggaatag aaagtatttg gttaggaaaa ccagtcttaa acataagata
     4501 tatttataaa agagtatcaa agaatgcaat acttacatct ccacttggtt attaacgagt
     4561 cgatgtccat gagcagggtg agcaactggt gtggttggct gaacctgggt tcatccctat
     4621 agaaggtgat catgatggct ccctgaaggg cacgatggct aaaccggcga tccccacgac
     4681 gcaccagtgc atcgtgcact gccggatcaa agatggagcg atacacctcg cgtcgcttct
     4741 caatgtccat gaggcggtag tttttcgcct tctccacggg ctcctccatg gcgctctgta
     4801 cctgcgcctc caggaatcga tcgacgctct cctgaaactt ggcccagaag ttgaagccac
     4861 tctcctccag tccgggcgtc ctctccagcc atcgctgcac tagctccagt agcgagggat
     4921 ctttctccga gttgcgaatc gagttccgcg cctcctcgtc gctaaagaca tccgagtact
     4981 tctggttgta tctcacccgc tgctctgtca gaactcccag cttgttctcg atcaaacgga
     5041 actgcagcga ctgaaaacca gatgcgggtg ccaggtactt gcggaagtcc atgaagtcta
     5101 gcggggtcat ggtctccaga atgggcactt ggtccaccag gagctgtaca aaggaagtta
     5161 taaacggatt ttggtaagag attcagaaag cactcacttt tagaatcaga accactcggt
     5221 tcagtcgctt gacaatctcc agcgtcttgg tttcatcgat gacctctgca tccaacatgt
     5281 ctcgtatgga gtcgaactca aagatgatct gcttgaacca aagctcgtag gctgtggcga
     5341 aggtacttaa atgccattga gtgttgtcat caaagttgta aacctactca ccctggtgcg
     5401 tgatgatgaa cagatgctca tcgtgcacgg gtcgcttgtc ctcctcggac agcatacact
     5461 gggcatccag cagtttgtcc agcatcagat actctccata gattttgccc acttccgtgg
     5521 ttaatggcac cgccgaatca tcgtgatcgt ttctgtatgg gtttgaattg aatcgcagaa
     5581 ctgaagatcg attggcattc ctggacagca cgtgctggtg ctcacccgtt tcctgcatag
     5641 ggacagctca tggtgcacag ctcagatcag atcgtgactc ctcgagcggc ggatgctggc
     5701 gaactgatct ccgccagcgg accggagatg agaccccagc gaaccgataa cagagcgaga
     5761 gagctccagt tccgactgat tgcacagtcg gtgatctggg cgatgggcac tgccagatag
     5821 gctgggaatt atcaatcact tgaggtgaaa gtgcggcgca cacaaatcca agcttgatat
     5881 cgaattccct gcagggcagt ttgtttggca tcatctcggc catcgtctta ttcttcttcg
     5941 tggcctttgt taccggagcg gctatgttgg gcgcggtgga gcccggaagc tgaaaggtta
     6001 atcgaatgcg ttaacttttc tgcaactcga aagtttgccg cctttgttcg actgccaata
     6061 actgttgatt cgaaaattcg aatcgaagcg gttgaatttc gtagggtggc caactacacc
     6121 aaagttcgcc ggcggtgtca tgcctgtttt tttttttttg ttcgcaatga ggaatggctc
     6181 ttaaaatcta ctagataaaa aaaatattca ttatttctat gctgctggaa cgcttcatta
     6241 atcttaaaaa ttctaa
//
