LOCUS Exported 7045 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION gRNA expression in B. burgdorferi, parental vector. ACCESSION . VERSION . KEYWORDS pBSV2_Psyn-sgRNA500 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7045) AUTHORS Takacs CN, Scott M, Chang Y, Kloos ZA, Irnov I, Rosa PA, Liu J, Jacobs-Wagner C TITLE A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi. JOURNAL Appl Environ Microbiol. 2020 Nov 30. pii: AEM.02519-20. doi: 10.1128/AEM.02519-20. PUBMED 33257311 REFERENCE 2 (bases 1 to 7045) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7045 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 71..659 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 560..579 /label=pBR322ori-F /note="pBR322 origin, forward primer" protein_bind 4142..4163 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4178..4208 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4216..4232 /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)." primer_bind 4221..4243 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4240..4256 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4240..4256 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 4293..4327 /label=J23119(SpeI) promoter /note="bacterial promoter (Registry of Standard Biological Parts BBa_J23119) modified to end with an SpeI site" misc_RNA 4844..4919 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" primer_bind complement(4962..4979) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4962..4978) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4971..4993) /label=M13/pUC Forward /note="In lacZ gene" CDS 5535..6344 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(5602..5621) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" regulatory 6598..6607 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 6604..6978 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" ORIGIN 1 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 61 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 121 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 181 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 241 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 301 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 361 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 421 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 481 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 541 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 601 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 661 acgccagcaa cgcggccttt ttacggttcc tgggcttttg ctggcctttt gctcacatgt 721 tctttcctgc actagatccc cggcaagttc atccttaggg tttccatatc tcttaaaaag 781 tcaatttctg cttgattaat ttcttgtgga ttattgtttt tgcggttttt cattttttat 841 ctccttgagt attgaatttt ttcttatata taaatatata gcaaaaacta tttttgccaa 901 cttttttaca aaaaattttt tattaaaaca cttagggctt tgctaaattc tcttttaaaa 961 gaacttagta aagccctatg gatttaagaa ctgattatac ttacgtagta aaaatactac 1021 agatattaat agtatagacc atatcaaaat cataatcaat ttatatttat ctaaaattgt 1081 agtccataca aaatacaaaa aaattaggtt tttctaaatt ctattttaaa agaacttagt 1141 aacacctaat ctctaaatta cttacaaaat tctaaaatta gatttttagt tcttcatatt 1201 tctttaaaag ttttttaaga aaatcttttt gattttcaaa aatttcatgc atcataaaac 1261 tagtaaattt ggattttttt ttgtaaaaat cataactttc ttgggtttta agttgaaatc 1321 ttaatggttt tattggattt tgcctagatt tttttaacgc cggactttct ttatctttta 1381 taaaattata agaattttta ataccatttt ctattaaata atcttctttt ataagcccct 1441 cttgcagtgc agttgctaat cttaaatact tatacgcttg agtttttgct acatcaaagt 1501 cttttataaa atcaagaaaa gttttataac catcaaattt ataatattga ttatctttta 1561 tttcttttaa aattttaata ttttccaccc tgtggaatat ttcttttttg aaattgtcat 1621 ttaacttttc ctttagtgtc ttgtaataag aaattttact ttcattaatc attaaatttt 1681 gttcaacata agtatctatc ttttttgtta aaactttaac aacacgtttt ttttccataa 1741 aaattttcct ttatttttgc aacattttcc ttaattgata taattgttac tattttagat 1801 tttttaatat ccacttaaga attaatccct cactagaata ttttttatta tagaattcat 1861 atacatgttt ttctaaatct ataaatttat caacaattgc tttattatag ggtgtatcta 1921 tcttttcttt tcttaataat cgtctcatat tctttaaata acaacaaaca ctgccggttt 1981 taaacctaaa ttctatgtaa tatatttttg aaaaagtata tacattttca accccgttta 2041 tttcatactt tacaaaaaca ttatttattg gttttctata accatagtaa atgcctaaaa 2101 atttgtcccc gtctttcata ggatatagat aaagtttttg aatctctgat ttatctaata 2161 atttttcaaa aaaaattata caccgctttt gtttgttact aactactcta tgcaatgcct 2221 ttaatatttt tgtgtgatat atatttttat cacctttttt ttctttcagc aggaagatac 2281 tttcttttgg aaatttaatt ttttgttcat ttattcttgc agccttaagt ccgattttca 2341 tatttaatcc ttatatagcc tttctattta attcttttaa actcaataaa cttttttggt 2401 ttttcattag ttctaataat tcgcaataat atgtgcaaaa cactttattg tattctattt 2461 tcttttggtt attcaaatat tcttttataa ttggctttag aagttcaata tttacttctt 2521 tttttagttg ttcaataagg atattgaaaa tgtttgtttt taaatcttga taattttgtt 2581 gagaattttc tttttttctt tcaattgact tctctaattt acgctttata ttgtttaaat 2641 cgctatattt atgactttca ataataaaat gaggcttaaa tttgtaattt tcgtatattt 2701 tttggaaact tgtttctaat tgtttagaat tatattcatt tttttcaaat tctttttgag 2761 tgttgcatag aatttccttt aatttttctt gtttttcttt catacaagat ttagtaaaat 2821 gtatattttt attttttagt aatttaattt ctattatttt tatgatttta agtattttga 2881 ttatattatc tttatcaata tttaaattga ataaaattgt aagaaaaatt gaaagaattt 2941 ttttacataa aaagttacat ttattgaagt aatttcttac ttgatacttt tctatttcat 3001 taatctttct ttcttctttt atattatttt tattatttaa acactccttt aaatctacac 3061 tcccattttt agtaaatcta tctttaaggc cccttgaaac tctattttga aatcttaaat 3121 cttttttttc ttgaaagtat ttatagattt tttggtaaca ttcttttttt gaaaaattaa 3181 gttgatagta tatttcggtc cccaaattta cccctaagtg tttgtgataa ttagttgtga 3241 ctttaaattc tttttctaat ttgtaaaggt aattttgcag tgtttttagt ttaacgggtg 3301 tttgactatt tcttttaagg ttttcattaa agtaatagag tatgtttttt tgtgtgtatt 3361 tcttatatgt tttatttatg tacgctagtg ttgagataag aactactaat ttatactgta 3421 atttattgta gcaagttgtg gttttactgg ttttgtttat attagataag ttttccattt 3481 atagctcctt gaataccgga ttttttatct ataaagtata tatatcacaa ttttgattta 3541 aaagtaaatt ctttttttaa aaatacaaaa attttttata aattatatag accatgatat 3601 tgtattaata ctaattgatt ttaatttaag gttgaactaa aataaacata gttttgtaga 3661 aaataatttt tcattatttt gctacttgat attggatcgt aaaaatatta gggctttaca 3721 aagttctttt aaaagagaat ttagtaaagc cctaagtctt ttaataaaaa attttttgta 3781 aaaaagttgg caaaaatagt ttttgctata tatttatata taagaaaaaa ttcaatactc 3841 aaggagataa aaaatggaaa acttaaacaa taatccacaa aaaattaatc aaataaataa 3901 gcaaactaat tttattggtt ttgaaaattt tatagaactt gataatcaag ttaatcaaac 3961 taaagaaaaa agtaaattaa gtaagatagg taagaaatta cccggaataa gcagtcaagt 4021 ctagtctagt gcggccgcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4081 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4141 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4201 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4261 attacgaatt cgagctcgaa ttctaaagat ctttgacagc tagctcagtc ctaggtataa 4321 tactagtgga agagccaaag tcaacactta tagacaaata tgatctttct aaccttcacg 4381 aaatagcatc agggggagca cctttgtcta aggaagtagg agaggcagtt gcaaagaggt 4441 ttcatttacc cggaattagg caaggatatg ggttaacaga aaccacatct gcaattttaa 4501 ttacacctga aggagatgat aaaccaggag ctgttgggaa ggttgttcct ttttttgagg 4561 ctaaagttgt tgatttagat actggaaaga ctttaggagt taatcaaaga ggcgagttat 4621 gtgtaagagg acctatgata atgtcaggat acgtaaataa tccagaagct acaaatgcat 4681 tgatagataa agatggttgg ttacactcag gcgatatagc ttattgggac gaagatgaac 4741 acttttttat agttgataga ttgaaatctt tgattaagta taaaggttat caagtagctc 4801 cagcagaatt ggaaagtatt ttattgcaac acccagctct tccgttttag agctagaaat 4861 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 4921 ttttttgaag cttgggcccg aacaaaaact caaagcttgg cactggccgt cgttttacaa 4981 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 5041 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 5101 agcctgaatg gcgaatggcg cggccgctac ccgagcttca aggaagattt cctattaagg 5161 ttgaacttaa gagcttaagc atagacgatt tgaaaaaaat tttaaaacaa acaaaaaatt 5221 ctttaataaa acaatatgtt gcgatgttta aggtttatga tttagattta aagtttagtg 5281 aggaagctat agatagaatt gcagagctta cttttaatat gaatcttgag agtgaaaatc 5341 ttggtgccag aagacttcac ggtgttatgg aaatagtgct tgcagatctt ttttttgaag 5401 tgcctggcag taagttgaaa aaatttgaaa taaacttgga ctatgttaat aaaaaaatac 5461 aaattaacga acaaaaagat ttgaattatt atataattta gttaaaagca attttaaatg 5521 agggaggttt ccatatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca 5581 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg 5641 cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca 5701 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat 5761 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca 5821 ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg 5881 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta 5941 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata 6001 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag 6061 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg 6121 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg 6181 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg 6241 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata 6301 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt 6361 ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcgctt acaatttcct 6421 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac aggtggcact 6481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 6541 tatccgctca tgagacaata accctgataa atgcttcaat aatagcacgt gaggagggcc 6601 accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg 6661 gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc 6721 ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg 6781 gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg 6841 gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag 6901 cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg 6961 gccgaggagc aggactgaca cgtgctaaaa cttcattttt aatttaaaag gatctaggtg 7021 aagatccttt ttgataatct catga //