LOCUS       sequence-275229-        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        1..400
                     /label=dU6-2 promoter
                     /note="RNA polymerase III promoter for Drosophila U6-2 
                     snRNA (Das et al., 1987)"
     misc_RNA        422..497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(582..602)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(582..600)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(621..637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(621..637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(634..656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    645..661
                     /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(669..675,676..693,694..699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    714..735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(852..869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1023..1611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1103..1122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1782..2573,2574..2642))
                     /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     2405..2424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2643..2747)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2773..3228)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2910..2931)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3122..3141
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3355..3377
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3369..3386
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3370..3386
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3396..3415
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3396..3414
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3622..3691
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             complement(join(3891..4008,4085..4178,4270..4876,4931..5064,
                     5125..5285,5359..5384))
                     /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 gttcgacttg cagcctgaaa tacggcacga gtaggaaaag ccgagtcaaa tgccgaatgc
       61 agagtctcat tacagcacaa tcaactcaag aaaaactcga cactttttta ccatttgcac
      121 ttaaatcctt ttttattcgt tatgtatact ttttttggtc cctaaccaaa acaaaaccaa
      181 actctcttag tcgtgcctct atatttaaaa ctatcaattt attatagtca ataaatcgaa
      241 ctgtgttttc aacaaacgaa caataggaca ctttgattct aaaggaaatt ttgaaaatct
      301 taagcagagg gttcttaaga ccatttgcca attcttataa ttctcaactg ctctttcctg
      361 atgttgatca tttatatagg tatgttttcc tcaatacttc gggtcttcga gttgaagacc
      421 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      481 tggcaccgag tcggtgcttt ttttgcggcc gcgcatgcac ttggctttcc accgttggta
      541 tcgattctct gggacgatga gtcgagctcc agcttttgtt ccctttagtg agggttaatt
      601 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
      661 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
      721 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
      781 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
      841 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
      901 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      961 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     1021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     1081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     1141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     1201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     1261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     1321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     1381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     1441 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     1501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     1561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     1621 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     1681 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     1741 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     1801 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1861 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1921 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1981 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2041 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2101 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2161 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2221 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2281 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2341 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     2401 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     2461 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     2521 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     2581 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     2641 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     2701 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     2761 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2821 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2881 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2941 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3001 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3061 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3121 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3181 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
     3241 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3301 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     3361 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
     3421 tgggtacctc gacatgcccg ccgtgaccgt cgagaacccg ctgacgctgc cccgcgtatc
     3481 cgcacccgcc gacgccgtcg cacgtcccgt gctcaccgtg accaccgcgc ccagcggttt
     3541 cgagggcgag ggcttcccgg tgcgccgcgc gttcgccggg atcaactacc gccacctcga
     3601 cccgttcatc atgatggacc agatgggtga ggtggagtac gcgcccgggg agcccaaggg
     3661 cacgccctgg cacccgcacc gcggcttcga gaccgtgacc tacatcgtcg acaagcttgg
     3721 atttattttg ttatgttata tgtattatat gtcagacata aagaaaagga acacatcaaa
     3781 tgtgataaca aagactaaac aagtaatttt attacaccaa aacgacaaaa cagtaggcag
     3841 aacaaacaac gcatagccaa acattgacga attggatacc ctgccgattg tcagacactt
     3901 ttgttgatca gtttcttgcg aatggtctcg tccagcggtg gaatcgcctc gcggggaatc
     3961 agaaaagtgg acagattgaa cagatccaga aacaccttgt accgatcact gaaaccaaaa
     4021 aaaaacaaag ggagaacagt ttgagttcat tgatccccga tataatcaca tctgcgatga
     4081 tcacctgaga gtggagcgca gatattgata tccagacgag ccaccagtgc ccaactgttg
     4141 ggatccaatc atgcgttgca ccatgatcac gtgattgtct gcggcgggaa tagaaagtat
     4201 ttggttagga aaaccagtct taaacataag atatatttat aaaagagtat caaagaatgc
     4261 aatacttaca tctccacttg gttattaacg agtcgatgtc catgagcagg gtgagcaact
     4321 ggtgtggttg gctgaacctg ggttcatccc tatagaaggt gatcatgatg gctccctgaa
     4381 gggcacgatg gctaaaccgg cgatccccac gacgcaccag tgcatcgtgc actgccggat
     4441 caaagatgga gcgatacacc tcgcgtcgct tctcaatgtc catgaggcgg tagtttttcg
     4501 ccttctccac gggctcctcc atggcgctct gtacctgcgc ctccaggaat cgatcgacgc
     4561 tctcctgaaa cttggcccag aagttgaagc cactctcctc cagtccgggc gtcctctcca
     4621 gccatcgctg cactagctcc agtagcgagg gatctttctc cgagttgcga atcgagttcc
     4681 gcgcctcctc gtcgctaaag acatccgagt acttctggtt gtatctcacc cgctgctctg
     4741 tcagaactcc cagcttgttc tcgatcaaac ggaactgcag cgactgaaaa ccagatgcgg
     4801 gtgccaggta cttgcggaag tccatgaagt ctagcggggt catggtctcc agaatgggca
     4861 cttggtccac caggagctgt acaaaggaag ttataaacgg attttggtaa gagattcaga
     4921 aagcactcac ttttagaatc agaaccactc ggttcagtcg cttgacaatc tccagcgtct
     4981 tggtttcatc gatgacctct gcatccaaca tgtctcgtat ggagtcgaac tcaaagatga
     5041 tctgcttgaa ccaaagctcg taggctgtgg cgaaggtact taaatgccat tgagtgttgt
     5101 catcaaagtt gtaaacctac tcaccctggt gcgtgatgat gaacagatgc tcatcgtgca
     5161 cgggtcgctt gtcctcctcg gacagcatac actgggcatc cagcagtttg tccagcatca
     5221 gatactctcc atagattttg cccacttccg tggttaatgg caccgccgaa tcatcgtgat
     5281 cgtttctgta tgggtttgaa ttgaatcgca gaactgaaga tcgattggca ttcctggaca
     5341 gcacgtgctg gtgctcaccc gtttcctgca tagggacagc tcatggtgca cagctcagat
     5401 cagatcgtga ctcctcgagc ggcggatgct ggcgaactga tctccgccag cggaccggag
     5461 atgagacccc agcgaaccga taacagagcg agagagctcc agttccgact gattgcacag
     5521 tcggtgatct gggcgatggg cactgccaga taggctggga attatcaatc acttgaggtg
     5581 aaagtgcggc gcacacaaat ccaagcttga tatcgaattc cctgcag
//
