LOCUS       Exported                6354 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Destination vector for Gateway based on vector #394 from the 
            Tol2Kit, containing a gRNA scaffold under the control of a zebrafish
            U6 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    pDestTol2pA2-U6:gRNA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6354)
  AUTHORS   Ablain J, Durand EM, Yang S, Zhou Y, Zon LI
  TITLE     A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in 
            Zebrafish.
  JOURNAL   Dev Cell. 2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032.
  PUBMED    25752963
REFERENCE   2  (bases 1 to 6354)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6354
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        20..124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             125..985
                     /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     complement(343..362)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1156..1744
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1645..1664
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1898..1915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2032..2053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2068..2098
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2106..2122
                     /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)."
     primer_bind     2111..2133
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2130..2146
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2165..2185
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2167..2185
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2222..2238
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2222..2238
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     polyA_signal    2910..3044
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2947..2966)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3001..3020
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_RNA        3443..3518
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     3531..3547
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3555..3678
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3704..3734
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3788..4447
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3854..3873)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4789..5094
                     /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
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     4973..4992
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5135..5259)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5278..5294)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6190..6211
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
       61 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      121 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
      181 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
      241 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
      301 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
      361 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
      421 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
      481 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
      541 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
      601 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
      661 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
      721 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
      781 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
      841 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
      901 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
      961 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     1021 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1081 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1321 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1381 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1441 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1501 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1561 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1621 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1681 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1741 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1801 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1861 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1921 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     1981 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     2041 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     2101 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     2161 gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
     2221 ctctagaact agtggatctg ctgggcttgc tgaaggtagg gggtcaagaa ccagaggtgt
     2281 aaagtacttg agtaatttta cttgattact gtacttaagt attatttttg gggattttta
     2341 ctttacttga gtacaattaa aaatcaatac ttttactttt acttaattac atttttttag
     2401 aaaaaaaagt actttttact ccttacaatt ttatttacag tcaaaaagta cttatttttt
     2461 ggagatcact tcattctatt ttcccttgct attaccaaac caattgaatt gcgctgatgc
     2521 ccagtttaat ttaaatgtta tttattctgc ctatgaaaat cgttttcaca ttatatgaaa
     2581 ttggtcagac atgttcattg gtcctttgga agtgacgtca tgtcacatct attaccacaa
     2641 tgcacagcac cttgacctgg aaattaggga aattataaca gtcaatcagt ggaagaaaat
     2701 ggaggaagta tgtgattcat cagcagctgc gagcagcaca gtccaaaatc agccacagga
     2761 tcaagagcac ccgtggccgt atcttcgcag atctgatcta gaggatcata atcagccata
     2821 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2881 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2941 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3001 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcatcatc gatgcgtctt
     3061 ttgttctggt catcaaggag gggggaatgt tctgcgcatg cctgtggggg agggagaagg
     3121 acacgtcact gaaaacgtcc ctgcatcaca ccgagacacc caatcactca agccgagacc
     3181 agataatttt gcatatgctt taaagtttga aaaataccac ggtaaaccct cacacaaact
     3241 ctggatttga gatctttcag gttttatcag tttgcaggtt tatgtcacca tgatataggg
     3301 tcagacttga tctaagggag ctgaataagt ggtttagtca ctcaccacct cccaaaaaca
     3361 tacccagaag tccctggtat atatagctct ccctccagct cttggttcgg ctagcactcc
     3421 tcagtgacga ggagtcagta gcgttttaga gctagaaata gcaagttaaa ataaggctag
     3481 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttggtacc gtaaaacgac
     3541 ggccagtgaa ttatcaacta tgtataataa agttgaacga gaaacgtaaa atgatataaa
     3601 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     3661 acatatccag tcactatggc ggccgcatta ggcaccccca ggctttacac tttatgcttc
     3721 cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga
     3781 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     3841 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     3901 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     3961 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
     4021 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     4081 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     4141 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     4201 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     4261 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     4321 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     4381 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg
     4441 ggcgtaatct agaggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc
     4501 tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg
     4561 tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag
     4621 gcatatatga tgtcaatatc tccggttcgg taagcacaac catgcagaat gaagcccgtc
     4681 gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt
     4741 ttattgaaat gaacggctcc tttgccgacg agaacagggg ctggtgaaat gcagtttaag
     4801 gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt
     4861 attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat
     4921 aaagtccccc gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg
     4981 accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc
     5041 caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca
     5101 ggctccctta tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt
     5161 acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc
     5221 attttacgtt tctcgttcaa cttttctata caaagttgat agcttggcgt aatcatggtc
     5281 atagctgttt cctgctcgag ggcccatctg gcctgtgttt cagacaccag ggagtctctg
     5341 ctcacgtttc ctgctatttg cagcctctct atcaagacta atacacctct tcccgcatcg
     5401 gctgcctgtg agaggctttt cagcactgca ggattgcttt tcagccccaa aagagctagg
     5461 cttgacacta acaattttga gaatcagctt ctactgaagt taaatctgag gttttacaac
     5521 tttgagtagc gtgtactggc attagattgt ctgtcttata gtttgataat taaatacaaa
     5581 cagttctaaa gcaggataaa accttgtatg catttcattt aatgtttttt gagattaaaa
     5641 gcttaaacaa gaatctctag ttttctttct tgcttttact tttacttcct taatactcaa
     5701 gtacaatttt aatggagtac ttttttactt ttactcaagt aagattctag ccagatactt
     5761 ttacttttaa ttgagtaaaa ttttccctaa gtacttgtac tttcacttga gtaaaatttt
     5821 tgagtacttt ttacacctct gtcaagaact cctggacaaa cctctgacct gtgtggaaca
     5881 gagtggatat gggtgtctga acagatattc acgtcttttg cagatcagag ggcatttctg
     5941 gtgttgtgca atgacctggg tccaacaggt aggcgtactc gtatccaaga gccttgttgg
     6001 acaggaggta gtctccattc ctataaagag gaaggaatgg caccatgtag tagccgtcat
     6061 tgtggccaat tggtgcattg gtacgtgggt agatggaccg gggaggctga tgagttctta
     6121 gccagcgtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     6181 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     6241 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     6301 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtgg
//
