LOCUS       sequence-200862-        6352 bp    DNA     circular SYN 15-DEC-2025
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    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  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 6352)
  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: "Dev Cell. 
            2015 Mar 4. pii: S1534-5807(15)00075-1. doi: 
            10.1016/j.devcel.2015.01.032."
COMMENT     Sequence Label: pDestTol2pA2-U6:gRNA
FEATURES             Location/Qualifiers
     source          1..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..124
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(125..193,194..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        join(2068..2073,2074..2091,2092..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"
     misc_feature    join(2272..2283,2284..2471)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     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        join(3703..3708,3709..3726,3727..3733)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3787..4467
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(3853..3872)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4787..5092
                     /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     4971..4990
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5133..5256)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5276..5292)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(5665..5827,5828..5839)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     6188..6209
                     /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 atgccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1141 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1201 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1261 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 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 ggcaccccag gctttacact ttatgcttcc
     3721 ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg agctaaggaa
     3781 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     3841 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     3901 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     3961 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa
     4021 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     4081 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     4141 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     4201 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     4261 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     4321 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     4381 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcaggcgggg
     4441 cgtaatctag aggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg
     4501 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta
     4561 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc
     4621 atatatgatg tcaatatctc cggttcggta agcacaacca tgcagaatga agcccgtcgt
     4681 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt
     4741 attgaaatga acggctcctt tgccgacgag aacaggggct ggtgaaatgc agtttaaggt
     4801 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     4861 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa
     4921 agtcccccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     4981 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     5041 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     5101 ctcccttata cacagccagt ctgcaggtcg accatagtga ctggatatgt tgtgttttac
     5161 agtattatgt agtctgtttt ttatgcaaaa tctaatttaa tatattgata tttatatcat
     5221 tttacgtttc tcgttcaact tttctataca aagttgatag cttggcgtaa tcatggtcat
     5281 agctgtttcc tgctcgaggg cccatctggc ctgtgtttca gacaccaggg agtctctgct
     5341 cacgtttcct gctatttgca gcctctctat caagactaat acacctcttc ccgcatcggc
     5401 tgcctgtgag aggcttttca gcactgcagg attgcttttc agccccaaaa gagctaggct
     5461 tgacactaac aattttgaga atcagcttct actgaagtta aatctgaggt tttacaactt
     5521 tgagtagcgt gtactggcat tagattgtct gtcttatagt ttgataatta aatacaaaca
     5581 gttctaaagc aggataaaac cttgtatgca tttcatttaa tgttttttga gattaaaagc
     5641 ttaaacaaga atctctagtt ttctttcttg cttttacttt tacttcctta atactcaagt
     5701 acaattttaa tggagtactt ttttactttt actcaagtaa gattctagcc agatactttt
     5761 acttttaatt gagtaaaatt ttccctaagt acttgtactt tcacttgagt aaaatttttg
     5821 agtacttttt acacctctgt caagaactcc tggacaaacc tctgacctgt gtggaacaga
     5881 gtggatatgg gtgtctgaac agatattcac gtcttttgca gatcagaggg catttctggt
     5941 gttgtgcaat gacctgggtc caacaggtag gcgtactcgt atccaagagc attgttggac
     6001 aggaggtagt ctccattcct ataaagagga aggaatggca ccatgtagta gccgtcattg
     6061 tggccaattg gtgcattggt acgtgggtag atggaccggg gaggctgatg agttcttagc
     6121 cagcgtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     6181 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     6241 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     6301 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt gg
//
