LOCUS       sequence-267245-        5627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression vector for Drosophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5627)
  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 5627)
  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-U6.2
FEATURES             Location/Qualifiers
     source          1..5627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..419
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        441..516
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(601..621)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(601..619)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(640..656)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(640..656)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(653..675)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    664..680
                     /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(688..694,695..712,713..718))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    733..754
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(871..888)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1042..1630)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1122..1141)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1801..2592,2593..2661))
                     /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     2424..2443
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2662..2766)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2792..3247)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2929..2950)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3141..3160
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3374..3396
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3388..3405
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3389..3405
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3415..3434
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3415..3433
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3641..3710
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             complement(join(3910..4027,4104..4197,4289..4895,4950..5083,
                     5144..5304,5378..5403))
                     /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 gatatcgaat tccctgcagg ttcgacttgc agcctgaaat acggcacgag taggaaaagc
       61 cgagtcaaat gccgaatgca gagtctcatt acagcacaat caactcaaga aaaactcgac
      121 acttttttac catttgcact taaatccttt tttattcgtt atgtatactt tttttggtcc
      181 ctaaccaaaa caaaaccaaa ctctcttagt cgtgcctcta tatttaaaac tatcaattta
      241 ttatagtcaa taaatcgaac tgtgttttca acaaacgaac aataggacac tttgattcta
      301 aaggaaattt tgaaaatctt aagcagaggg ttcttaagac catttgccaa ttcttataat
      361 tctcaactgc tctttcctga tgttgatcat ttatataggt atgttttcct caatacttcg
      421 ggtcttcgag ttgaagacct gttttagagc tagaaatagc aagttaaaat aaggctagtc
      481 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttgcggccg cgcatgcact
      541 tggctttcca ccgttggtat cgattctctg ggacgatgag tcgagctcca gcttttgttc
      601 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
      661 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
      721 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
      781 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
      841 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
      901 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
      961 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1021 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1081 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1141 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1201 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1261 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1321 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1381 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1441 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     1501 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1561 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1621 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1681 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1741 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     1801 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     1861 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     1921 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     1981 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2041 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2101 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2161 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2221 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2281 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2341 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2401 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2461 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2521 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2581 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2641 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2701 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     2761 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     2821 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2881 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2941 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3001 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3061 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     3121 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     3181 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     3241 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3301 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     3361 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
     3421 gactcactat agggcgaatt gggtacctcg acatgcccgc cgtgaccgtc gagaacccgc
     3481 tgacgctgcc ccgcgtatcc gcacccgccg acgccgtcgc acgtcccgtg ctcaccgtga
     3541 ccaccgcgcc cagcggtttc gagggcgagg gcttcccggt gcgccgcgcg ttcgccggga
     3601 tcaactaccg ccacctcgac ccgttcatca tgatggacca gatgggtgag gtggagtacg
     3661 cgcccgggga gcccaagggc acgccctggc acccgcaccg cggcttcgag accgtgacct
     3721 acatcgtcga caagcttgga tttattttgt tatgttatat gtattatatg tcagacataa
     3781 agaaaaggaa cacatcaaat gtgataacaa agactaaaca agtaatttta ttacaccaaa
     3841 acgacaaaac agtaggcaga acaaacaacg catagccaaa cattgacgaa ttggataccc
     3901 tgccgattgt cagacacttt tgttgatcag tttcttgcga atggtctcgt ccagcggtgg
     3961 aatcgcctcg cggggaatca gaaaagtgga cagattgaac agatccagaa acaccttgta
     4021 ccgatcactg aaaccaaaaa aaaacaaagg gagaacagtt tgagttcatt gatccccgat
     4081 ataatcacat ctgcgatgat cacctgagag tggagcgcag atattgatat ccagacgagc
     4141 caccagtgcc caactgttgg gatccaatca tgcgttgcac catgatcacg tgattgtctg
     4201 cggcgggaat agaaagtatt tggttaggaa aaccagtctt aaacataaga tatatttata
     4261 aaagagtatc aaagaatgca atacttacat ctccacttgg ttattaacga gtcgatgtcc
     4321 atgagcaggg tgagcaactg gtgtggttgg ctgaacctgg gttcatccct atagaaggtg
     4381 atcatgatgg ctccctgaag ggcacgatgg ctaaaccggc gatccccacg acgcaccagt
     4441 gcatcgtgca ctgccggatc aaagatggag cgatacacct cgcgtcgctt ctcaatgtcc
     4501 atgaggcggt agtttttcgc cttctccacg ggctcctcca tggcgctctg tacctgcgcc
     4561 tccaggaatc gatcgacgct ctcctgaaac ttggcccaga agttgaagcc actctcctcc
     4621 agtccgggcg tcctctccag ccatcgctgc actagctcca gtagcgaggg atctttctcc
     4681 gagttgcgaa tcgagttccg cgcctcctcg tcgctaaaga catccgagta cttctggttg
     4741 tatctcaccc gctgctctgt cagaactccc agcttgttct cgatcaaacg gaactgcagc
     4801 gactgaaaac cagatgcggg tgccaggtac ttgcggaagt ccatgaagtc tagcggggtc
     4861 atggtctcca gaatgggcac ttggtccacc aggagctgta caaaggaagt tataaacgga
     4921 ttttggtaag agattcagaa agcactcact tttagaatca gaaccactcg gttcagtcgc
     4981 ttgacaatct ccagcgtctt ggtttcatcg atgacctctg catccaacat gtctcgtatg
     5041 gagtcgaact caaagatgat ctgcttgaac caaagctcgt aggctgtggc gaaggtactt
     5101 aaatgccatt gagtgttgtc atcaaagttg taaacctact caccctggtg cgtgatgatg
     5161 aacagatgct catcgtgcac gggtcgcttg tcctcctcgg acagcataca ctgggcatcc
     5221 agcagtttgt ccagcatcag atactctcca tagattttgc ccacttccgt ggttaatggc
     5281 accgccgaat catcgtgatc gtttctgtat gggtttgaat tgaatcgcag aactgaagat
     5341 cgattggcat tcctggacag cacgtgctgg tgctcacccg tttcctgcat agggacagct
     5401 catggtgcac agctcagatc agatcgtgac tcctcgagcg gcggatgctg gcgaactgat
     5461 ctccgccagc ggaccggaga tgagacccca gcgaaccgat aacagagcga gagagctcca
     5521 gttccgactg attgcacagt cggtgatctg ggcgatgggc actgccagat aggctgggaa
     5581 ttatcaatca cttgaggtga aagtgcggcg cacacaaatc caagctt
//
