LOCUS       sequence-317621-        9332 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA expression in B. burgdorferi, parental vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9332)
  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 9332)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl 
            Environ Microbiol. 2020 Nov 30. pii: AEM.02519-20. doi: 
            10.1128/AEM.02519-20."
COMMENT     Sequence Label: pBSV2H_Psyn-sgRNA500
FEATURES             Location/Qualifiers
     source          1..9332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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    4136..4157
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4172..4177,4178..4195,4196..4202)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4210..4226
                     /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     4215..4237
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4234..4250
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4234..4250
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4286..4320
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        4837..4912
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4955..4972)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4955..4971)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4964..4986)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             6178..7197
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     CDS             8281..9300
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
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 atactgttct 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 tggccttttg ctggcctttt gctcacatgt
      721 tctttcctgc actagatccc cggcaagttc atccttaggg ttttcatatc tcttaaaaag
      781 tcaatttctg cttgattaat ttcttgtgga ttattgtttt tgcggttttt cattttttat
      841 ctccttgagt attgaatttt ttcttatata taaaatatag caaaaactat ttttgccaac
      901 ttttttacaa aaaatttttt attaaaacac ttagggcttt gctaaattct cttttaaaag
      961 aacttagtaa agccctatgg atttaagaac tgattatact tacgtagtaa aaatactaca
     1021 gatattaata gtatagacca tatcaaaatc ataatcaatt tatatttatc taaaattgtg
     1081 gtccatacaa aatacaaaaa aattaggttt ttctaaattc tattttaaaa gaacttagta
     1141 acacctaatc tctaaattac ttacaaaatt ctaaaattag atttttagtt cttcatattt
     1201 ctttaaaagt tttttaagaa aatctttttg attttcaaaa atttcatgca tcataaaact
     1261 agtaaatttg gatttttttt tgtaaaaatc ataactttct tgggttttaa gttgaaatct
     1321 taatggtttt attggatttt gcctagattt ttttaacgcc ggactttctt tatcttttat
     1381 aaaattataa gaatttttaa taccattttc tattaaataa tcttctttta taagcccctc
     1441 ttgcagtgca gttgctaatc ttaaatactt atacgcttga gtttttgcta catcaaagtc
     1501 ttttataaaa tcaagaaaag ttttataacc atcaaattta taatattgat tatcttttat
     1561 ttcttttaaa attttaatat tttccaccct gtggaatatt tcttttttga aattgtcatt
     1621 taacttttcc tttagtgtct tgtaataaga aattttactt tcattaatca ttaaattttg
     1681 ttcaacataa gtatctatct tttttgttaa aactttaaca acacgttttt tttccataaa
     1741 aattttcctt tatttttgca acattttcct taattgatat aattgttact attttagatt
     1801 ttttaatatc cacttaagaa ttaatccctc actagaatat tttttattat agaattcata
     1861 tacatgtttt tctaaatcta taaatttatc aacaattgct ttattatagg gtgtatctat
     1921 cttttctttt cttaataatc gtctcatatt ctttaaataa caacaaacac tgccggtttt
     1981 aaacctaaat tctatgtaat atatttttga aaaagtatat acattttcaa ccccgtttat
     2041 ttcatacttt acaaaaacat tatttattgg ttttctataa ccatagtaaa tgcctaaaaa
     2101 tttgtccccg tctttcatag gatatagata aagtttttga atctctgatt tatctaataa
     2161 tttttcaaaa aaaattatac accgcttttg tttgttacta actactctat gcaatgcctt
     2221 taatattttt gtgtgatata tatttttatc accttttttt tctttcagca ggaagatact
     2281 ttcttttgga aatttaattt tttgttcatt tattcttgca gccttaagtc cgattttcat
     2341 atttaatcct tatatagcct ttctatttaa ttcttttaaa ctcaataaac ttttttggtt
     2401 tttcattagt tctaataatt cgcaataata tgtgcaaaac actttattgt attctatttt
     2461 cttttggtta ttcaaatatt cttttataat tggctttaga agttcaatat ttacttcttt
     2521 ttttagttgt tcaataagga tattgaaaat gtttgttttt aaatcttgat aattttgttg
     2581 agaattttct ttttttcttt caattgactt ctctaattta cgctttatat tgtttaaatc
     2641 gctatattta tgactttcaa taataaaatg aggcttaaat ttgtaatttt cgtatatttt
     2701 ttggaaactt gtttctaatt gtttagaatt atattcattt ttttcaaatt ctttttgagt
     2761 gttgcataga atttccttta atttttcttg tttttctttc atacaagatt tagtaaaatg
     2821 tatattttta ttttttagta atttaatttc tattattttt atgattttaa gtattttgat
     2881 tatattatct ttatcaatat ttaaattgaa taaaattgta agaaaaattg aaagaatttt
     2941 tttacataaa aagttacatt tattgaagta atttcttact tgatactttt ctatttcatt
     3001 aatctttctt tcttctttta tattattttt attatttaaa cactccttta aatctacact
     3061 cccattttta gtaaatctat ctttaaggcc ccttgaaact ctattttgaa atcttaaatc
     3121 ttttttttct tgaaagtatt tatagatttt ttggtaacat tctttttttg aaaaattaag
     3181 ttgatagtat atttcggtcc ccaaatttac ccctaagtgt ttgtgataat tagttgtgac
     3241 tttaaattct ttttctaatt tgtaaaggta attttgcagt gtttttagtt taacgggtgt
     3301 ttgactattt cttttaaggt tttcattaaa gtaatagagt atgttttttt gtgtgtattt
     3361 cttatatgtt ttatttatgt acgctagtgt tgagataagg actactaatt tatactgtaa
     3421 tttattgtag caagttgtgg ttttactggt tttgtttata ttagataagt tttccattta
     3481 tagctccttg aataccggat tttttatcta taaagtatat atatcacaat tttgatttaa
     3541 aagtaaattc tttttttaaa aatacaaaaa ttttttataa attatataga ccatgatatt
     3601 gtattaatac taattgattt taatttaagg ttgaactaaa ataaacatag ttttgtagaa
     3661 aataattttt cattattttg ctacttgata ttggatcgta aaaatattag ggctttacaa
     3721 agttctttta aaagagaatt tagtaaagcc ctaagtcttt taataaaaaa ttttttgtaa
     3781 aaaagttggc aaaaatagtt tttgctatat atttatatat aagaaaaaat tcaatactca
     3841 aggagataaa aaatggaaaa cttaaacaat aatccacaaa aaattaatca aataaataag
     3901 caaactaatt ttattggttt tgaaaatttt atagaacttg ataatcaagt taatcaaact
     3961 aaagaaaaaa gtaaattaag taagataggt aagaaattac ccggaataag cagtcaagtc
     4021 tagtgcggcc gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     4081 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     4141 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     4201 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     4261 attcgagctc gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt
     4321 ggaagagcca aagtcaacac ttatagacaa atatgatctt tctaaccttc acgaaatagc
     4381 atcaggggga gcacctttgt ctaaggaagt aggagaggca gttgcaaaga ggtttcattt
     4441 acccggaatt aggcaaggat atgggttaac agaaaccaca tctgcaattt taattacacc
     4501 tgaaggagat gataaaccag gagctgttgg gaaggttgtt cctttttttg aggctaaagt
     4561 tgttgattta gatactggaa agactttagg agttaatcaa agaggcgagt tatgtgtaag
     4621 aggacctatg ataatgtcag gatacgtaaa taatccagaa gctacaaatg cattgataga
     4681 taaagatggt tggttacact caggcgatat agcttattgg gacgaagatg aacacttttt
     4741 tatagttgat agattgaaat ctttgattaa gtataaaggt tatcaagtag ctccagcaga
     4801 attggaaagt attttattgc aacacccagc tcttccgttt tagagctaga aatagcaagt
     4861 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg
     4921 aagcttgggc ccgaacaaaa actcaaagct tggcactggc cgtcgtttta caacgtcgtg
     4981 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     5041 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     5101 atggcgaatg gcgcggccgc cctaggttaa tcttcaataa catgtaaacc acgacgtttt
     5161 aaatcttcta atgaatctaa cattcctctt attaattcaa caggatgtcc ttcccaatct
     5221 tcaacaacac caacaattct taaaggttca caagttctat aagattgtgt tggattacca
     5281 ggaaattttt tatttgtaag atttggatca tcttcaaatg gtcctgttgg ttcaactata
     5341 tatatataac cacgaccttc aagaccagat agtgacatag caagttcagc tccccaaact
     5401 gctggttcca ttaaagctga aaaataaata tgtttaagaa ttcgaccatc ttcaaaatga
     5461 gaaataaatc ctgtagttag taaatcacca attgctaaat tagctttagt tccatgataa
     5521 aatggtcctt gtacttgttt ataattatca tgagaaatag gaatccaatc ttttaccatg
     5581 acgttacctc ctcatgatgt ttaactttgt tttagggcga ctgccctgct gcgtaacatc
     5641 gttgctgctc cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc
     5701 ccgaggcata gactgtaccc caaaaaaaca gtcataacaa gccatgcggc cgtacccgag
     5761 cttcaaggaa gatttcctat taaggttgaa cttaagagct taagcataga cgatttgaaa
     5821 aaaattttaa aacaaacaaa aaattcttta ataaaacaat atgttgcgat gtttaaggtt
     5881 tatgatttag atttaaagtt tagtgaggaa gctatagata gaattgcaga gcttactttt
     5941 aatatgaatc ttgagagtga aaatcttggt gccagaagac ttcacggtgt tatggaaata
     6001 gtgcttgcag atcttttttt tgaagtgcct ggcagtaagt tgaaaaaatt tgaaataaac
     6061 ttggactatg ttaataaaaa aatacaaatt aacgaacaaa aagatttgaa ttattatata
     6121 atttagttaa aagcaatttt aaatgaggga ggtttccatc atatggatag aagtggtaaa
     6181 cctgaattaa ctgctactag tgttgaaaaa tttttaattg aaaaatttga tagtgttagt
     6241 gatttaatgc aattaagtga aggtgaagaa agtagagctt ttagttttga tgttggtggt
     6301 agaggttatg ttttaagagt taatagttgt gctgatggtt tttataaaga tagatatgtt
     6361 tatagacatt ttgctagtgc tgctttacct attcctgaag ttttagatat tggtgaattt
     6421 agtgaaagtt taacttattg tattagtaga agagcacaag gtgttacttt acaagattta
     6481 cctgaaactg aattacctgc tgttttacaa cctgttgctg aagctatgga tgctattgcg
     6541 gcggctgatc tcagtcaaac ttcgggtttt ggtccttttg gtcctcaagg tattggtcaa
     6601 tatactactt ggagagattt tatttgtgct attgctgatc ctcatgttta tcattggcaa
     6661 actgttatgg atgatactgt tagtgctagt gttgctcaag cattagatga attaatgtta
     6721 tgggctgaag attgtcctga agttagacat ttagttcatg ctgattttgg tagtaataat
     6781 gttttaactg ataatggtag aattactgct gttattgatt ggagtgaagc tatgtttggt
     6841 gatagtcaat atgaagttgc taatattttt ttttggagac cttggttagc ttgtatggaa
     6901 caacaaacta gatattttga aagaagacat cctgaattag ctggtagtcc tagattaaga
     6961 gcttatatgt taagaattgg tttagatcaa ttatatcaaa gtttagttga tggtaatttt
     7021 gatgatgctg cttgggctca aggtagatgt gatgctattg ttagaagtgg tgctggtact
     7081 gttggtagaa ctcaaattgc tagaagaagt gctgctgttt ggactgatgg ttgtgttgaa
     7141 gttttagctg atagtggtaa tagaagacct agtactagac ctagagctaa agaataacaa
     7201 ttcgttcaag ccgagatcgg cttacgcggt taatcttcaa taacatgtaa accacgacgt
     7261 tttaaatctt ctaatgaatc taacattcct cttattaatt caacaggatg tccttcccaa
     7321 tcttcaacaa caccaacaat tcttaaaggt tcacaagttc tataagattg tgttggatta
     7381 ccaggaaatt ttttatttgt aagatttgga tcatcttcaa atggtcctgt tggttcaact
     7441 atatatatat aaccacgacc ttcaagacca gatagtgaca tagcaagttc agctccccaa
     7501 actgctggtt ccattaaagc tgaaaaataa atatgtttaa gaattcgacc atcttcaaaa
     7561 tgagaaataa atcctgtagt tagtaaatca ccaattgcta aattagcttt agttccatga
     7621 taaaatggtc cttgtacttg tttataatta tcatgagaaa taggaatcca atcttttacc
     7681 atgacgttac ctcctcatga tgtttaactt tgttttaggg cgactgccct gctgcgtaac
     7741 atcgttgctg ctccataaca tcaaacatcg acccacggcg taacgcgctt gctgcttgga
     7801 tgcccgaggc atagactgta ccccaaaaaa acagtcataa caagccatgc ggccgtaccc
     7861 gagcttcaag gaagatttcc tattaaggtt gaacttaaga gcttaagcat agacgatttg
     7921 aaaaaaattt taaaacaaac aaaaaattct ttaataaaac aatatgttgc gatgtttaag
     7981 gtttatgatt tagatttaaa gtttagtgag gaagctatag atagaattgc agagcttact
     8041 tttaatatga atcttgagag tgaaaatctt ggtgccagaa gacttcacgg tgttatggaa
     8101 atagtgcttg cagatctttt ttttgaagtg cctggcagta agttgaaaaa atttgaaata
     8161 aacttggact atgttaataa aaaaatacaa attaacgaac aaaaagattt gaattattat
     8221 ataatttagt taaaagcaat tttaaatgag ggaggtttcc atcatatgga tagaagtggt
     8281 aaacctgaat taactgctac tagtgttgaa aaatttttaa ttgaaaaatt tgatagtgtt
     8341 agtgatttaa tgcaattaag tgaaggtgaa gaaagtagag cttttagttt tgatgttggt
     8401 ggtagaggtt atgttttaag agttaatagt tgtgctgatg gtttttataa agatagatat
     8461 gtttatagac attttgctag tgctgcttta cctattcctg aagttttaga tattggtgaa
     8521 tttagtgaaa gtttaactta ttgtattagt agaagagcac aaggtgttac tttacaagat
     8581 ttacctgaaa ctgaattacc tgctgtttta caacctgttg ctgaagctat ggatgctatt
     8641 gcggcggctg atctcagtca aacttcgggt tttggtcctt ttggtcctca aggtattggt
     8701 caatatacta cttggagaga ttttatttgt gctattgctg atcctcatgt ttatcattgg
     8761 caaactgtta tggatgatac tgttagtgct agtgttgctc aagcattaga tgaattaatg
     8821 ttatgggctg aagattgtcc tgaagttaga catttagttc atgctgattt tggtagtaat
     8881 aatgttttaa ctgataatgg tagaattact gctgttattg attggagtga agctatgttt
     8941 ggtgatagtc aatatgaagt tgctaatatt tttttttgga gaccttggtt agcttgtatg
     9001 gaacaacaaa ctagatattt tgaaagaaga catcctgaat tagctggtag tcctagatta
     9061 agagcttata tgttaagaat tggtttagat caattatatc aaagtttagt tgatggtaat
     9121 tttgatgatg ctgcttgggc tcaaggtaga tgtgatgcta ttgttagaag tggtgctggt
     9181 actgttggta gaactcaaat tgctagaaga agtgctgctg tttggactga tggttgtgtt
     9241 gaagttttag ctgatagtgg taatagaaga cctagtacta gacctagagc taaagaataa
     9301 caattcgttc aagccgagat cggcttacgc gt
//
