LOCUS       sequence-107698-        6809 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  destination vector for one U6x:sgRNA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6809)
  AUTHORS   Yin L, Maddison LA, Li M, Kara N, LaFave MC, Varshney GK, Burgess 
            SM, Patton JG, Chen W
  TITLE     Multiplex Conditional Mutagenesis Using Transgenic Expression of 
            Cas9 and sgRNAs.
  JOURNAL   Genetics. 2015 Apr 8. pii: genetics.115.176917.
  PUBMED    25855067
REFERENCE   2  (bases 1 to 6809)
  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: "Genetics. 
            2015 Apr 8. pii: genetics.115.176917."
COMMENT     Sequence Label: pGGDestTol2LC-1sgRNA
FEATURES             Location/Qualifiers
     source          1..6809
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(622..633,634..796)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     873..889
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    909..1032
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1073..1378)
                     /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     complement(1175..1194)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1720..2379)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2294..2313
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2433..2439,2440..2457,2458..2463))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2489..2612)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2620..2636)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(3230..3232,3233..3235,3236..3949)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3275..3296)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3536..3555)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3883..3904
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3967..3984)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3973..4180
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(4339..4526,4527..4538)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4572..4588)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4572..4588)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4625..4645)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4625..4643)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4664..4680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4677..4699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4688..4704
                     /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(4712..4718,4719..4736,4737..4742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4895..4912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5066..5654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5146..5165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5825..6616,6617..6685))
                     /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     6448..6467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6686..6790)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
      241 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
      301 ggagcccccg atttagagct tgacggggaa agccgggctg gctaagaact catcagcctc
      361 cccggtccat ctacccacgt accaatgcac caattggcca caatgacggc tactacatgg
      421 tgccattcct tcctctttat aggaatggag actacctcct gtccaacaag gctcttggat
      481 acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc
      541 tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga
      601 ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga
      661 aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc
      721 ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta
      781 aaagtaaaag caagaaagaa aactagagat tcttgtttaa gcttttaatc tcaaaaaaca
      841 ttaaatgaaa tgcatcagta gagaccctcg agcaggaaac agctatgacc atgattacgc
      901 caagctatca actttgtata gaaaagttga acgagaaacg taaaatgata taaatatcaa
      961 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
     1021 ccagtcacta tggtcgacct gcagactggc tgtgtataag ggagcctgac atttatattc
     1081 cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca
     1141 cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
     1201 tttcatcccc gatatgcacc accgggtaaa gttcacgggg gactttatct gacagcagac
     1261 gtgcactggc cagggggatc accatccgtc gcccgggcgt gtcaataata tcactctgta
     1321 catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta aactgcattt
     1381 caccagcccc tgttctcgtc ggcaaaagag ccgttcattt caataaaccg ggcgacctca
     1441 gccatccctt cctgattttc cgctttccag cgttcggcac gcagacgacg ggcttcattc
     1501 tgcatggttg tgcttaccga accggagata ttgacatcat atatgccttg agcaactgat
     1561 agctgtcgct gtcaactgtc actgtaatac gctgcttcat agcatacctc tttttgacat
     1621 acttcgggta tacatatcag tatatattct tataccgcaa aaatcagcgc gcaaatacgc
     1681 atactgttat ctggctttta gtaagccgga tcctctagat tacgccccgc cctgccactc
     1741 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     1801 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     1861 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     1921 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     1981 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     2041 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     2101 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     2161 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     2221 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     2281 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     2341 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     2401 aaatctcgac ggatcctaac tcaaaatcca cacattatac gagccggaag cataaagtgt
     2461 aaagcctggg ggtgcctaat gcggccgcca tagtgactgg atatgttgtg ttttacagta
     2521 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     2581 cgtttctcgt tcaactttat tatacatagt tgataattca ctggccgtcg ttttacggta
     2641 cgctagcggt ctctagtcgg taccattaat agtgtgcatt cagtgcagga tgttatgtat
     2701 ttattttctg tagtgcaaac tttttcaaag aattttccag tctaagcttt tcagtcaaga
     2761 aaaaaaaaaa gcgattgatt atctgtgata tttaaaaaaa ttagctctta aatctggtga
     2821 accaagtggc cacaacacat taaatactga cgttctacag ccatgtagct tcatctggtc
     2881 tggtttgttt tggcaggcac ttattttccg gtgatagttg gccacaagag tattgtctga
     2941 gccattcagt gctagactgt cattctcagg tcagagtcca ccccgctgat tctgctgaca
     3001 acactgttcc caccatgaga taatgccatt ccagagagat ccatttgtaa gcccctcttt
     3061 ctgcagcaca ggtatataac caggggtctg cctccactaa ggccggcaca catcatttgg
     3121 ggatctttgt actgtactag cctctattag cctcctactt gcgttcagtt cacatctttt
     3181 gttcatcagg tgcaataagt tacagtaagg agttaccagg tctgacaaca tggtgagcaa
     3241 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3301 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3361 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     3421 cctgacctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt
     3481 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     3541 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     3601 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     3661 caacgccatc agcgacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc
     3721 caacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     3781 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     3841 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     3901 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggcccgct
     3961 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     4021 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     4081 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     4141 agggggagga ttgggaagac aatagcaggc atgctgggga gagatcgccg ccacccactt
     4201 ccaaaggacc aatgaacatg tctgaccaat ttcatataat gtgaaaacga ttttcatagg
     4261 cagaataaat aacatttaaa ttaaactggg catcagcgca attcaattgg tttggtaata
     4321 gcaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa
     4381 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag
     4441 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca
     4501 gtaatcaagt aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag
     4561 caagcccagc agatccacta gttctagagc ggccgccacc gcggtggagc tccagctttt
     4621 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     4681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     5521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5941 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     6001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6781 gggttccgcg cacatttccc cgaaaagtg
//
