LOCUS Exported 6423 bp ds-DNA circular SYN 21-MAR-2016 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pCas35 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6423) AUTHORS . TITLE Direct Submission JOURNAL Exported Monday, Mar 21, 2016 from SnapGene 3.1.1 http://www.snapgene.com COMMENT LOCUS pUC19 2686 bp DNA circular 18-OCT-2007 DEFINITION Cloning vector pUC19, complete sequence. ACCESSION VERSION KEYWORDS . SOURCE Cloning vector pUC19 ORGANISM Cloning vector pUC19 other sequences; artificial sequences; vectors. REFERENCE 1 (bases 1 to 2686) AUTHORS New England Biolabs. TITLE Direct Submission JOURNAL Submitted (18-OCT-2007) Research Department, New England Biolabs, 240 County Road, Ipswich, MA 01938, USA COMMENT See also GenBank accession L09137. FEATURES Location/Qualifiers source 1..6423 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(146..397) /codon_start=1 /note="beta-galactosidase alpha fragment" /translation="SLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQ LRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA" misc_feature 397..1276 /note="NS1 Top" gene 397..1014 /note="SYNPCC7002_A0935" misc_feature 1278..1298 /note="tetR Insert Fwd" misc_feature 1337..1377 /note="T7 terminator" gene complement(1413..1449) /note="ssRa" gene complement(1450..2069) /note="tetR" RBS 2076..2087 /note="RBS" misc_feature 2096..2130 /note="J23113 Promoter" misc_feature complement(2112..2150) /note="TetR" /note="TetR (TetO2*) Ins Rev" misc_feature 2132..2205 /note="Promoter" misc_feature 2152..2168 /note="TetO1" -10_signal 2169..2174 /note="-35" misc_feature 2175..2191 /note="TetO1" -10_signal 2192..2198 /note="-10" misc_feature 2199 /note="TSS from EZ3" misc_feature 2206..2225 /note="sgRNA cpcB" misc_feature 2206..2225 /note="gBlock NS1 sgRNA YFP" misc_feature 2226..2267 /note="cas9 Handle" misc_feature 2268..2308 /note="S. pyogenes terminator" misc_feature 2309..2409 /note="Spacer Included in Qi et. al. 2013" /note="Spacer Included in Qi et. al. 2013 ""Repurposing CRISPR...""" terminator 2410..2675 /note="rrnB_terminator" misc_feature 2676..3676 /note="KanR Cassette" CDS 2777..3592 /note="KanR" misc_feature 3678..4188 /note="NS1 Bottom" gene complement(3768..4160) /note="SYNPCC7002_A0936" misc_feature 4189..4213 /note="Untitled" rep_origin complement(4604..5192) /direction=LEFT /note="pMB1 origin of replication" /note="pMB1 origin of replication (counter-clockwise) (RNAII -35 to RNA/DNA switch point)" misc_RNA complement(4604..5156) /note="RNAII transcript (complementary strand)" -35_signal 5010..5015 /note="RNAI promoter (clockwise); TTGAAG" -10_signal 5032..5037 /note="RNAI promoter (clockwise); GCTACA" misc_RNA 5046..5153 /note="RNAI transcript" -10_signal complement(5166..5171) /note="RNAII promoter" /note="RNAII promoter (counter-clockwise); CGTAAT" -35_signal complement(5187..5192) /note="RNAII promoter" /note="RNAII promoter (counter-clockwise); TTGAGA" CDS complement(5363..6223) /codon_start=1 /note="beta-lactamase" /note="ampR (confers resistance to ampicillin)" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" -10_signal complement(6267..6272) /note="bla promoter (counter-clockwise); GAGACA" -35_signal complement(6288..6293) /note="bla promoter (counter-clockwise); TTCAAA" ORIGIN 1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 361 tttcccagtc acgacgttgt aaaacgacgg ccagtgatgt ttggtcgaaa attcttcaca 421 cttttaacgg ctgtctccac agtgttcctg actcctattt acggagcggc tcaaacgatg 481 caagatcaaa gtaatacgat gaataatgaa aattcattgg ggaatagtgc gactggggta 541 gtcaaccaaa ccgctgtgta caacttaggt caaagtgcca cctacggttt tagagggatc 601 gtttgtcccc gcccttcctt tgtgctcagt ggctctgcaa atggcaatac atttgatggg 661 gagagttcaa attcctacgc tctttccggt gccattattg tgcccctcgg tggtcaagtg 721 ggcagcaact gtagagattt cagcactacc ctactggaaa gagaacaagc cgaattagca 781 aagtttcaaa tccaatcttc agcggaaaca gtcaaacttt gtgctgacat gatttcttca 841 ggcattcaag tagatccaga ggtttatcca gtactggcgc ggcaatgttc tgcagttcgg 901 ttggtttcac aaggacgggc ttctagcaca aatgaagccc gacaaaatga atcgactcac 961 aataagctag atgatgatgt agaattgtct gctaattact taaaaactga gtaaatataa 1021 tattttgaat agtgaggaaa aatatctcaa aggctatatg aaaataaaaa aagagcggct 1081 aaaaaatctt cagcttacag tgacgcctgt ctttgcattc taaagcacta aaacacagtc 1141 gtttttttac tgactccaag tgtttttatt actatactta gaaattcgtc aaataggcat 1201 gaaattgctg tattaaaata tcctactgta tcttttagtt cacactattt ttcttgagtt 1261 aattttgtat caataactat aaacgcagaa aggcccaccc gaaggtgagc cagtgtgact 1321 ctagtagaga gcgttcaacc ccttggggcc tctaaacggg tcttgagggg ttttttgtct 1381 agtagtgatc tacactagca ctatcagtgt tattaagcta ctaaagcgta gttttcgtcg 1441 tttgcagcgg acccactttc acatttaagt tgtttttcta atccgcatat gatcaattca 1501 aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat agcttgtcgt 1561 aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc gacttgatgc 1621 tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag tgcatataat 1681 gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc gagagtttca 1741 tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg atgacttagt 1801 aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct ttccccttct 1861 aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc gtcgagcaaa 1921 gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag cttctgggcg 1981 agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta 2041 atcactttac ttttatctaa tctggacatc tagtatttct cctctttctc tagtagctag 2101 cataatccct aggactgagc tagccatcag gttaacaaaa aagcaggaat atccctatca 2161 gtgatagaca gacatcccta tcagtgatag agtataaagt taactgctag aaatgaactc 2221 acctcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 2281 aaagtggcac cgagtcggtg ctttttttga agcttgggcc cgaacaaaaa ctcatctcag 2341 aagaggatct gaatagcgcc gtcgatcatc atcatcatca tcattgagtt taaacggtct 2401 ccagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga 2461 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc 2521 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc 2581 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggcccag tctttcgact 2641 gagcctttcg ttttatctgt tgtttgtcgg tgaactacgg ggtctgacgc tcagtggaac 2701 gaaaactcac gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag 2761 taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt 2821 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca 2881 atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa 2941 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct 3001 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg 3061 gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga 3121 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc 3181 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg 3241 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt 3301 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac 3361 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat 3421 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg 3481 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa 3541 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat 3601 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 3661 catttccccg aaaaggtgga tccgaaaata aagtcaaagt gcttctataa tttaagatct 3721 atttttaaac agtacagctt tacagaaaga aaaaccagaa gggacgccta ttttatctcg 3781 ttttcttccc agagtagcag gacatttttt tctgtaaaag cagagcaatt tgtactgaaa 3841 tcattatcaa taatgacatt agattcttcg atcaagacat ctgtaccgtt gcttatatct 3901 gcttcagagc ctaatacaat ggggtattgt ggttgttgac atgcagcatt aacgagtact 3961 ttgactggga atacaaccga atcatcgcta taggtgtaga cattggcgtg gtagtaatag 4021 gtatagtcat catagtcgta attatattca taaccatacc gatactggta atcaaaatca 4081 ataaatacag gttcactttt tgcctgttga acaacgataa atgatagacc actaagggct 4141 gctgtaaaca gtttattcat tttgctttct ccatcccttg acttaatttg gcgtaatcat 4201 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 4261 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 4441 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4501 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4561 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4621 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4681 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4741 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4801 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4861 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4921 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4981 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5041 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5101 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5161 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5221 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5281 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5341 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5401 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 5461 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 5521 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 5581 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 5641 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 5701 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 5761 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 5821 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 5881 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 5941 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6001 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6061 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6121 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6181 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6241 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6301 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 6361 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 6421 gtc //