LOCUS       sequence-177022-        8260 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Gateway based on vector #395 from the 
            Tol2Kit, containing a gRNA scaffold under the control of a zebrafish
            U6 promoter, and cmlc2:GFP as a transgenesis marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8260)
  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 8260)
  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: pDestTol2CG2-U6:gRNA
FEATURES             Location/Qualifiers
     source          1..8260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..121
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(122..190,191..982)
                     /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(340..359)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1153..1741
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1642..1661
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1895..1912
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2029..2050
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2065..2070,2071..2088,2089..2095)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2103..2119
                     /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     2108..2130
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2127..2143
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2162..2182
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2164..2182
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     2219..2235
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2219..2235
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(2269..2280,2281..2468)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     CDS             join(3690..3692,3693..3695,3696..4409)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3690..3692,3693..3695,3696..4406)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3735..3756)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3996..4015)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4343..4364
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4711..4845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_RNA        5244..5319
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     5332..5348
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5356..5479
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5504..5509,5510..5527,5528..5534)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5588..6268
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(5654..5673)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6588..6893
                     /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     6772..6791
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6934..7057)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7077..7093)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    join(7466..7628,7629..7640)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     8093..8114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
       61 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
      121 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
      181 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
      241 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
      301 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
      361 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
      421 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
      481 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
      541 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
      601 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
      661 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
      721 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
      781 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
      841 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
      901 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
      961 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     1021 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     1081 gaccaaaatg ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     1141 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     1201 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     1261 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     1321 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     1381 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     1441 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     1501 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1561 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1621 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1681 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1741 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1801 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     1861 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     1921 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1981 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     2041 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     2101 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     2161 cgcaattaac cctcactaaa gggaacaaaa gctggagctc caccgcggtg gcggccgctc
     2221 tagaactagt ggatctgctg ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa
     2281 gtacttgagt aattttactt gattactgta cttaagtatt atttttgggg atttttactt
     2341 tacttgagta caattaaaaa tcaatacttt tacttttact taattacatt tttttagaaa
     2401 aaaaagtact ttttactcct tacaatttta tttacagtca aaaagtactt attttttgga
     2461 gatcacttca ttctattttc ccttgctatt accaaaccaa ttgaattgcg ctgatgccca
     2521 gtttaattta aatgttattt attctgccta tgaaaatcgt tttcacatta tatgaaattg
     2581 gtcagacatg ttcattggtc ctttggaagt gacgtcatgt cacatctatt accacaatgc
     2641 acagcacctt gacctggaaa ttagggaaat tataacagtc aatcagtgga agaaaatgga
     2701 ggaagtatgt gattcatcag cagctgcgag cagcacagtc caaaatcagc cacaggatca
     2761 agagcacccg tggccgtatc ttcgcagatc taaagcttaa atcagttgtg ttaaataaga
     2821 gacattcaaa ataaatgtaa atgagctctc caaatcagca gacttaacat tctttaaaat
     2881 gattgattca atagtgataa aaatcaggca tagccagttg taactttaga taaattacag
     2941 aaaatgtcaa atacagagaa ccgattcttt tttatgatac atccaagcac acatttaaca
     3001 caatccaggc aaaccccgaa tttcacagtc acaagcactg tttgtacaag agctttgcct
     3061 aaggacacac agtctctata agtccaggtc gttggtttca ctcttatttt aaacatgtga
     3121 catttttcct gccatcctgt cttaggctgc tgtttgcttc attccatgtc acattaaatt
     3181 cctcagtagc accttttaca cacacagcca atcttttcca gaaaattcaa ttgctttgaa
     3241 gagataatgt gtgaacaaat ccatttagaa aaggaaaatt aagaatttgt aaaatcatct
     3301 gtaaattgtt ggcattcttc tgtatatgaa catcacatca tttacaggta aaggtctggt
     3361 cattaattat atgacaattt actggtatta ttttgtgaaa ggggctattt tcaatgcgtt
     3421 catccatcct tttcatccct caaatctctc attcacgtcc ccctccccat ctgcacactt
     3481 tatctcattt ccaccctgct ggaatctgag cacttgtgca gttatcaggg ctcctgtatt
     3541 taggaggctc tgggtgtcca tgtaggggac gaacagaaac actgcagacc tttatagaag
     3601 aacaattgat aagagtcctc atacataaag actccattag taagccagtg acccaggagc
     3661 ccagaccaac agcaaagcag acagtgacca tggtgagcaa gggcgaagag ctgttcaccg
     3721 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3781 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3841 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3901 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3961 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     4021 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     4081 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     4141 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     4201 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     4261 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     4321 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4381 ctctcggcat ggacgagctg tacaagtaaa gcggcccctc tcgagcctct agaactatag
     4441 tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg gacaaaccac
     4501 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4561 tgtaaccatt ataagctgca ataaacaaga gatctgatct agaggatcat aatcagccat
     4621 accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg
     4681 aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac
     4741 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4801 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatcatcat cgatgcgtct
     4861 tttgttctgg tcatcaagga ggggggaatg ttctgcgcat gcctgtgggg gagggagaag
     4921 gacacgtcac tgaaaacgtc cctgcatcac accgagacac ccaatcactc aagccgagac
     4981 cagataattt tgcatatgct ttaaagtttg aaaaatacca cggtaaaccc tcacacaaac
     5041 tctggatttg agatctttca ggttttatca gtttgcaggt ttatgtcacc atgatatagg
     5101 gtcagacttg atctaaggga gctgaataag tggtttagtc actcaccacc tcccaaaaac
     5161 atacccagaa gtccctggta tatatagctc tccctccagc tcttggttcg gctagcactc
     5221 ctcagtgacg aggagtcagt agcgttttag agctagaaat agcaagttaa aataaggcta
     5281 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttggtac cgtaaaacga
     5341 cggccagtga attatcaact atgtataata aagttgaacg agaaacgtaa aatgatataa
     5401 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     5461 aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc
     5521 cggctcgtat aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga
     5581 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     5641 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     5701 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     5761 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa
     5821 agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca
     5881 aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca
     5941 catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt
     6001 tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt
     6061 aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac
     6121 gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg
     6181 cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcaggcggg
     6241 gcgtaatcta gaggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct
     6301 gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt
     6361 atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg
     6421 catatatgat gtcaatatct ccggttcggt aagcacaacc atgcagaatg aagcccgtcg
     6481 tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt
     6541 tattgaaatg aacggctcct ttgccgacga gaacaggggc tggtgaaatg cagtttaagg
     6601 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
     6661 ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     6721 aagtcccccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     6781 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc
     6841 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     6901 gctcccttat acacagccag tctgcaggtc gaccatagtg actggatatg ttgtgtttta
     6961 cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca
     7021 ttttacgttt ctcgttcaac ttttctatac aaagttgata gcttggcgta atcatggtca
     7081 tagctgtttc ctgctcgagg gcccatctgg cctgtgtttc agacaccagg gagtctctgc
     7141 tcacgtttcc tgctatttgc agcctctcta tcaagactaa tacacctctt cccgcatcgg
     7201 ctgcctgtga gaggcttttc agcactgcag gattgctttt cagccccaaa agagctaggc
     7261 ttgacactaa caattttgag aatcagcttc tactgaagtt aaatctgagg ttttacaact
     7321 ttgagtagcg tgtactggca ttagattgtc tgtcttatag tttgataatt aaatacaaac
     7381 agttctaaag caggataaaa ccttgtatgc atttcattta atgttttttg agattaaaag
     7441 cttaaacaag aatctctagt tttctttctt gcttttactt ttacttcctt aatactcaag
     7501 tacaatttta atggagtact tttttacttt tactcaagta agattctagc cagatacttt
     7561 tacttttaat tgagtaaaat tttccctaag tacttgtact ttcacttgag taaaattttt
     7621 gagtactttt tacacctctg tcaagaactc ctggacaaac ctctgacctg tgtggaacag
     7681 agtggatatg ggtgtctgaa cagatattca cgtcttttgc agatcagagg gcatttctgg
     7741 tgttgtgcaa tgacctgggt ccaacaggta ggcgtactcg tatccaagag cattgttgga
     7801 caggaggtag tctccattcc tataaagagg aaggaatggc accatgtagt agccgtcatt
     7861 gtggccaatt ggtgcattgg tacgtgggta gatggaccgg ggaggctgat gagttcttag
     7921 ccagcctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     7981 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     8041 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     8101 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     8161 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     8221 aattttaaca aaatattaac gcttacaatt taggtggcac
//
