LOCUS       Exported                7037 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 7037)
  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 7037)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7037
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_RNA        517..592
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(635..652)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(635..651)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(644..666)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1208..2017
                     /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(1275..1294)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     regulatory      2268..2277
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2274..2648
                     /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"
     rep_origin      2786..3374
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3275..3294
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    6852..6873
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6888..6918
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6926..6942
                     /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     6931..6953
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6950..6966
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6950..6966
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7003..7037
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
ORIGIN
        1 ggaagagcca aagtcaacac ttatagacaa atatgatctt tctaaccttc acgaaatagc
       61 atcaggggga gcacctttgt ctaaggaagt aggagaggca gttgcaaaga ggtttcattt
      121 acccggaatt aggcaaggat atgggttaac agaaaccaca tctgcaattt taattacacc
      181 tgaaggagat gataaaccag gagctgttgg gaaggttgtt cctttttttg aggctaaagt
      241 tgttgattta gatactggaa agactttagg agttaatcaa agaggcgagt tatgtgtaag
      301 aggacctatg ataatgtcag gatacgtaaa taatccagaa gctacaaatg cattgataga
      361 taaagatggt tggttacact caggcgatat agcttattgg gacgaagatg aacacttttt
      421 tatagttgat agattgaaat ctttgattaa gtataaaggt tatcaagtag ctccagcaga
      481 attggaaagt attttattgc aacacccagc tcttccgttt tagagctaga aatagcaagt
      541 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
      601 aagcttgggc ccgaacaaaa actcaaagct tggcactggc cgtcgtttta caacgtcgtg
      661 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
      721 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
      781 atggcgaatg gcgcggccgc tacccgagct tcaaggaaga tttcctatta aggttgaact
      841 taagagctta agcatagacg atttgaaaaa aattttaaaa caaacaaaaa attctttaat
      901 aaaacaatat gttgcgatgt ttaaggttta tgatttagat ttaaagttta gtgaggaagc
      961 tatagataga attgcagagc ttacttttaa tatgaatctt gagagtgaaa atcttggtgc
     1021 cagaagactt cacggtgtta tggaaatagt gcttgcagat cttttttttg aagtgcctgg
     1081 cagtaagttg aaaaaatttg aaataaactt ggactatgtt aataaaaaaa tacaaattaa
     1141 cgaacaaaaa gatttgaatt attatataat ttagttaaaa gcaattttaa atgagggagg
     1201 tttccatatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     1261 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     1321 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     1381 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     1441 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     1501 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     1561 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     1621 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     1681 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     1741 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     1801 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     1861 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     1921 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     1981 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     2041 acactggcag agcattacgc tgacttgacg ggacggcgct tacaatttcc tgatgcggta
     2101 ttttctcctt acgcatctgt gcggtatttc acaccgcatc aggtggcact tttcggggaa
     2161 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2221 tgagacaata accctgataa atgcttcaat aatagcacgt gaggagggcc accatggcca
     2281 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
     2341 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
     2401 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
     2461 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
     2521 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
     2581 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
     2641 aggactgaca cgtgctaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2701 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2761 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2821 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2881 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2941 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3001 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3061 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3121 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3181 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3241 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3301 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3361 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3421 cttttgctca catgttcttt cctgcactag atccccggca agttcatcct tagggttttc
     3481 atatctctta aaaagtcaat ttctgcttga ttaatttctt gtggattatt gtttttgcgg
     3541 tttttcattt tttatctcct tgagtattga attttttctt atatataaat atatagcaaa
     3601 aactattttt gccaactttt ttacaaaaaa ttttttatta aaacacttag ggctttgcta
     3661 aattctcttt taaaagaact tagtaaagcc ctatggattt aagaactgat tatacttacg
     3721 tagtaaaaat actacagata ttaatagtat agaccatatc aaaatcataa tcaatttata
     3781 tttatctaaa attgtagtcc atacaaaata caaaaaaatt aggtttttct aaattctatt
     3841 ttaaaagaac ttagtaacac ctaatctcta aattacttac aaaattctaa aattagattt
     3901 ttagttcttc atatttcttt aaaagttttt taagaaaatc tttttgattt tcaaaaattt
     3961 catgcatcat aaaactagta aatttggatt tttttttgta aaaatcataa ctttcttggg
     4021 ttttaagttg aaatcttaat ggttttattg gattttgcct agattttttt aacgccggac
     4081 tttctttatc ttttataaaa ttataagaat ttttaatacc attttctatt aaataatctt
     4141 cttttataag cccctcttgc agtgcagttg ctaatcttaa atacttatac gcttgagttt
     4201 ttgctacatc aaagtctttt ataaaatcaa gaaaagtttt ataaccatca aatttataat
     4261 attgattatc ttttatttct tttaaaattt taatattttc caccctgtgg aatatttctt
     4321 ttttgaaatt gtcatttaac ttttccttta gtgtcttgta ataagaaatt ttactttcat
     4381 taatcattaa attttgttca acataagtat ctatcttttt tgttaaaact ttaacaacac
     4441 gttttttttc cataaaaatt ttcctttatt tttgcaacat tttccttaat tgatataatt
     4501 gttactattt tagatttttt aatatccact taagaattaa tccctcacta gaatattttt
     4561 tattatagaa ttcatataca tgtttttcta aatctataaa tttatcaaca attgctttat
     4621 tatagggtgt atctatcttt tcttttctta ataatcgtct catattcttt aaataacaac
     4681 aaacactgcc ggttttaaac ctaaattcta tgtaatatat ttttgaaaaa gtatatacat
     4741 tttcaacccc gtttatttca tactttacaa aaacattatt tattggtttt ctataaccat
     4801 agtaaatgcc taaaaatttg tccccgtctt tcataggata tagataaagt ttttgaatct
     4861 ctgatttatc taataatttt tcaaaaaaaa ttatacaccg cttttgtttg ttactaacta
     4921 ctctatgcaa tgcctttaat atttttgtgt gatatatatt tttatcacct tttttttctt
     4981 tcagcaggaa gatactttct tttggaaatt taattttttg ttcatttatt cttgcagcct
     5041 taagtccgat tttcatattt aatccttata tagcctttct atttaattct tttaaactca
     5101 ataaactttt ttggtttttc attagttcta ataattcgca ataatatgtg caaaacactt
     5161 tattgtattc tattttcttt tggttattca aatattcttt tataattggc tttagaagtt
     5221 caatatttac ttcttttttt agttgttcaa taaggatatt gaaaatgttt gtttttaaat
     5281 cttgataatt ttgttgagaa ttttcttttt ttctttcaat tgacttctct aatttacgct
     5341 ttatattgtt taaatcgcta tatttatgac tttcaataat aaaatgaggc ttaaatttgt
     5401 aattttcgta tattttttgg aaacttgttt ctaattgttt agaattatat tcattttttt
     5461 caaattcttt ttgagtgttg catagaattt cctttaattt ttcttgtttt tctttcatac
     5521 aagatttagt aaaatgtata tttttatttt ttagtaattt aatttctatt atttttatga
     5581 ttttaagtat tttgattata ttatctttat caatatttaa attgaataaa attgtaagaa
     5641 aaattgaaag aattttttta cataaaaagt tacatttatt gaagtaattt cttacttgat
     5701 acttttctat ttcattaatc tttctttctt cttttatatt atttttatta tttaaacact
     5761 cctttaaatc tacactccca tttttagtaa atctatcttt aaggcccctt gaaactctat
     5821 tttgaaatct taaatctttt ttttcttgaa agtatttata gattttttgg taacattctt
     5881 tttttgaaaa attaagttga tagtatattt cggtccccaa atttacccct aagtgtttgt
     5941 gataattagt tgtgacttta aattcttttt ctaatttgta aaggtaattt tgcagtgttt
     6001 ttagtttaac gggtgtttga ctatttcttt taaggttttc attaaagtaa tagagtatgt
     6061 ttttttgtgt gtatttctta tatgttttat ttatgtacgc tagtgttgag ataaggacta
     6121 ctaatttata ctgtaattta ttgtagcaag ttgtggtttt actggttttg tttatattag
     6181 ataagttttc catttatagc tccttgaata ccggattttt tatctataaa gtatatatat
     6241 cacaattttg atttaaaagt aaattctttt tttaaaaata caaaaatttt ttataaatta
     6301 tatagaccat gatattgtat taatactaat tgattttaat ttaaggttga actaaaataa
     6361 acatagtttt gtagaaaata atttttcatt attttgctac ttgatattgg atcgtaaaaa
     6421 tattagggct ttacaaagtt cttttaaaag agaatttagt aaagccctaa gtcttttaat
     6481 aaaaaatttt ttgtaaaaaa gttggcaaaa atagtttttg ctatatattt atatataaga
     6541 aaaaattcaa tactcaagga gataaaaaat ggaaaactta aacaataatc cacaaaaaat
     6601 taatcaaata aataagcaaa ctaattttat tggttttgaa aattttatag aacttgataa
     6661 tcaagttaat caaactaaag aaaaaagtaa attaagtaag ataggtaaga aattacccgg
     6721 aataagcagt caagtctagt gcggccgcgc ccaatacgca aaccgcctct ccccgcgcgt
     6781 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6841 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     6901 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6961 tatgaccatg attacgaatt cgagctcgaa ttctaaagat ctttgacagc tagctcagtc
     7021 ctaggtataa tactagt
//
