LOCUS       sequence-107699-        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  destination vector for two U6x:sgRNA cassettes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6806)
  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 6806)
  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-2sgRNA
FEATURES             Location/Qualifiers
     source          1..6806
                     /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(3227..3229,3230..3232,3233..3946)
                     /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(3272..3293)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3533..3552)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3880..3901
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3964..3981)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3970..4177
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(4336..4523,4524..4535)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4569..4585)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4569..4585)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4622..4642)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4622..4640)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4661..4677)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4674..4696)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4685..4701
                     /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(4709..4715,4716..4733,4734..4739))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4754..4775
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4892..4909)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5063..5651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5143..5162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5822..6613,6614..6682))
                     /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     6445..6464
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6683..6787)
                     /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 cgctaggtct ctctcagtac cattaatagt gtgcattcag tgcaggatgt tatgtattta
     2701 ttttctgtag tgcaaacttt ttcaaagaat tttccagtct aagcttttca gtcaagaaaa
     2761 aaaaaaagcg attgattatc tgtgatattt aaaaaaatta gctcttaaat ctggtgaacc
     2821 aagtggccac aacacattaa atactgacgt tctacagcca tgtagcttca tctggtctgg
     2881 tttgttttgg caggcactta ttttccggtg atagttggcc acaagagtat tgtctgagcc
     2941 attcagtgct agactgtcat tctcaggtca gagtccaccc cgctgattct gctgacaaca
     3001 ctgttcccac catgagataa tgccattcca gagagatcca tttgtaagcc cctctttctg
     3061 cagcacaggt atataaccag gggtctgcct ccactaaggc cggcacacat catttgggga
     3121 tctttgtact gtactagcct ctattagcct cctacttgcg ttcagttcac atcttttgtt
     3181 catcaggtgc aataagttac agtaaggagt taccaggtct gacaacatgg tgagcaaggg
     3241 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     3301 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     3361 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     3421 gacctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     3481 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     3541 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     3601 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     3661 cgccatcagc gacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     3721 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     3781 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     3841 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     3901 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg gcccgctgat
     3961 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4021 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4081 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4141 gggaggattg ggaagacaat agcaggcatg ctggggagag atcgccgcca cccacttcca
     4201 aaggaccaat gaacatgtct gaccaatttc atataatgtg aaaacgattt tcataggcag
     4261 aataaataac atttaaatta aactgggcat cagcgcaatt caattggttt ggtaatagca
     4321 agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt aaataaaatt
     4381 gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa gtaaaagtat
     4441 tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt aagtacagta
     4501 atcaagtaaa attactcaag tactttacac ctctggttct tgacccccta ccttcagcaa
     4561 gcccagcaga tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt
     4621 ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4681 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4741 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4801 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4861 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4921 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4981 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5041 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5101 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5161 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5221 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5281 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5341 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5401 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5461 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5521 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5581 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5641 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5701 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5761 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5821 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5881 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5941 ccagtgctgc aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa
     6001 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6061 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6121 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6181 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6241 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6301 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6361 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6421 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6481 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6541 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6601 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6661 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6721 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6781 ttccgcgcac atttccccga aaagtg
//
