LOCUS       sequence-107700-        6805 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  destination vector for three U6x:sgRNA cassettes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6805)
  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 6805)
  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-3sgRNA
FEATURES             Location/Qualifiers
     source          1..6805
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(143..164)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(621..632,633..795)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     872..888
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    908..1031
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1072..1377)
                     /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(1174..1193)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1719..2378)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2293..2312
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(2432..2438,2439..2456,2457..2462))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(2488..2611)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2619..2635)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(3226..3228,3229..3231,3232..3945)
                     /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(3271..3292)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3532..3551)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3879..3900
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3963..3980)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3969..4176
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    join(4335..4522,4523..4534)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(4568..4584)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4568..4584)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4621..4641)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4621..4639)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4660..4676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4673..4695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4684..4700
                     /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(4708..4714,4715..4732,4733..4738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4753..4774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4891..4908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5062..5650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5142..5161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5821..6612,6613..6681))
                     /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     6444..6463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6682..6786)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
       61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
      121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
      181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
      241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
      301 gagcccccga tttagagctt gacggggaaa gccgggctgg ctaagaactc atcagcctcc
      361 ccggtccatc tacccacgta ccaatgcacc aattggccac aatgacggct actacatggt
      421 gccattcctt cctctttata ggaatggaga ctacctcctg tccaacaagg ctcttggata
      481 cgagtacgcc tacctgttgg acccaggtca ttgcacaaca ccagaaatgc cctctgatct
      541 gcaaaagacg tgaatatctg ttcagacacc catatccact ctgttccaca caggtcagag
      601 gtttgtccag gagttcttga cagaggtgta aaaagtactc aaaaatttta ctcaagtgaa
      661 agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg gctagaatct
      721 tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt aaggaagtaa
      781 aagtaaaagc aagaaagaaa actagagatt cttgtttaag cttttaatct caaaaaacat
      841 taaatgaaat gcatcagtag agaccctcga gcaggaaaca gctatgacca tgattacgcc
      901 aagctatcaa ctttgtatag aaaagttgaa cgagaaacgt aaaatgatat aaatatcaat
      961 atattaaatt agattttgca taaaaaacag actacataat actgtaaaac acaacatatc
     1021 cagtcactat ggtcgacctg cagactggct gtgtataagg gagcctgaca tttatattcc
     1081 ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac
     1141 ttcttccccg ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct
     1201 ttcatccccg atatgcacca ccgggtaaag ttcacggggg actttatctg acagcagacg
     1261 tgcactggcc agggggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac
     1321 atccacaaac agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc
     1381 accagcccct gttctcgtcg gcaaaagagc cgttcatttc aataaaccgg gcgacctcag
     1441 ccatcccttc ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct
     1501 gcatggttgt gcttaccgaa ccggagatat tgacatcata tatgccttga gcaactgata
     1561 gctgtcgctg tcaactgtca ctgtaatacg ctgcttcata gcatacctct ttttgacata
     1621 cttcgggtat acatatcagt atatattctt ataccgcaaa aatcagcgcg caaatacgca
     1681 tactgttatc tggcttttag taagccggat cctctagatt acgccccgcc ctgccactca
     1741 tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca
     1801 tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc
     1861 atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg
     1921 aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa
     1981 taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg
     2041 aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg
     2101 gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg
     2161 aattccggat gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg
     2221 tgcttatttt tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta
     2281 taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat
     2341 atatcaacgg tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa
     2401 aatctcgacg gatcctaact caaaatccac acattatacg agccggaagc ataaagtgta
     2461 aagcctgggg gtgcctaatg cggccgccat agtgactgga tatgttgtgt tttacagtat
     2521 tatgtagtct gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac
     2581 gtttctcgtt caactttatt atacatagtt gataattcac tggccgtcgt tttacggtac
     2641 gctaggtctc tccaagtacc attaatagtg tgcattcagt gcaggatgtt atgtatttat
     2701 tttctgtagt gcaaactttt tcaaagaatt ttccagtcta agcttttcag tcaagaaaaa
     2761 aaaaaagcga ttgattatct gtgatattta aaaaaattag ctcttaaatc tggtgaacca
     2821 agtggccaca acacattaaa tactgacgtt ctacagccat gtagcttcat ctggtctggt
     2881 ttgttttggc aggcacttat tttccggtga tagttggcca caagagtatt gtctgagcca
     2941 ttcagtgcta gactgtcatt ctcaggtcag agtccacccc gctgattctg ctgacaacac
     3001 tgttcccacc atgagataat gccattccag agagatccat ttgtaagccc ctctttctgc
     3061 agcacaggta tataaccagg ggtctgcctc cactaaggcc ggcacacatc atttggggat
     3121 ctttgtactg tactagcctc tattagcctc ctacttgcgt tcagttcaca tcttttgttc
     3181 atcaggtgca ataagttaca gtaaggagtt accaggtctg acaacatggt gagcaagggc
     3241 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3301 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3361 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3421 acctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     3481 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3541 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3601 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3661 gccatcagcg acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac
     3721 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3781 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3841 tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3901 accgccgccg ggatcactct cggcatggac gagctgtaca agtaaagcgg cccgctgatc
     3961 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     4021 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     4081 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     4141 ggaggattgg gaagacaata gcaggcatgc tggggagaga tcgccgccac ccacttccaa
     4201 aggaccaatg aacatgtctg accaatttca tataatgtga aaacgatttt cataggcaga
     4261 ataaataaca tttaaattaa actgggcatc agcgcaattc aattggtttg gtaatagcaa
     4321 gggaaaatag aatgaagtga tctccaaaaa ataagtactt tttgactgta aataaaattg
     4381 taaggagtaa aaagtacttt tttttctaaa aaaatgtaat taagtaaaag taaaagtatt
     4441 gatttttaat tgtactcaag taaagtaaaa atccccaaaa ataatactta agtacagtaa
     4501 tcaagtaaaa ttactcaagt actttacacc tctggttctt gaccccctac cttcagcaag
     4561 cccagcagat ccactagttc tagagcggcc gccaccgcgg tggagctcca gcttttgttc
     4621 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4681 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4741 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4801 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4861 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5221 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5461 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5641 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5701 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5761 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5941 cagtgctgca atgataccgc gggacccacg ctcaccggct ccagatttat cagcaataaa
     6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6121 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6661 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6721 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6781 tccgcgcaca tttccccgaa aagtg
//
