LOCUS       Exported                6736 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  gRNA expression in B. burgdorferi, parental vector.
ACCESSION   .
VERSION     .
KEYWORDS    pKFSS1_Psyn-sgRNA500
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  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 6736)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6736
                     /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             5545..6336
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
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 cggccgccct aggttaatac ccgagcttca aggaagattt
     5161 cctattaagg ttgaacttaa gagcttaagc atagacgatt tgaaaaaaat tttaaaacaa
     5221 acaaaaaatt ctttaataaa acaatatgtt gcgatgttta aggtttatga tttagattta
     5281 aagtttagtg aggaagctat agatagaatt gcagagctta cttttaatat gaatcttgag
     5341 agtgaaaatc ttggtgccag aagacttcac ggtgttatgg aaatagtgct tgcagatctt
     5401 ttttttgaag tgcctggcag taagttgaaa aaatttgaaa taaacttgga ctatgttaat
     5461 aaaaaaatac aaattaacga acaaaaagat ttgaattatt atataattta gttaaaagca
     5521 attttaaatg agggaggttt ccatatgagg gaagcggtga tcgccgaagt atcgactcaa
     5581 ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat
     5641 ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt
     5701 acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa
     5761 acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg
     5821 cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
     5881 cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct
     5941 atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa
     6001 ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta
     6061 tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
     6121 atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca
     6181 atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt
     6241 ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac
     6301 gtgaaaggcg agatcaccaa ggtagtcggc aaataagacg tcgccggagc ggtcgagttc
     6361 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     6421 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     6481 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     6541 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     6601 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     6661 caggactgac acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6721 tttgataatc tcatga
//
