LOCUS Exported 6806 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION destination vector for four U6x:sgRNA cassettes. ACCESSION . VERSION . KEYWORDS pGGDestTol2LC-4sgRNA 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6806 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(144..165) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 873..889 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 908..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(2433..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 3227..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" 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(4709..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(5822..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 ctcctggtac 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 //