LOCUS       sequence-306457-        6731 bp    DNA     circular SYN 15-APR-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 6731)
  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 6731)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pKFSS1_Psyn-sgRNA500
FEATURES             Location/Qualifiers
     source          1..6731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    354..375
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(390..395,396..413,414..420)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    428..444
                     /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     433..455
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     452..468
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     452..468
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        504..538
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1055..1130
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1173..1190)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1173..1189)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1182..1204)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1756..2547
                     /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"
     rep_origin      3019..3607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3508..3527
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagttggcaa aaatagtttt tgctatatat ttatatataa gaaaaaattc aatactcaag
       61 gagataaaaa atggaaaact taaacaataa tccacaaaaa attaatcaaa taaataagca
      121 aactaatttt attggttttg aaaattttat agaacttgat aatcaagtta atcaaactaa
      181 agaaaaaagt aaattaagta agataggtaa gaaattaccc ggaataagca gtcaagtcta
      241 gtgcggccgc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
      301 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
      361 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
      421 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacat gattacgaat
      481 tcgagctcga attctaaaga tctttgacag ctagctcagt cctaggtata atactagtgg
      541 aagagccaaa gtcaacactt atagacaaat atgatctttc taaccttcac gaaatagcat
      601 cagggggagc acctttgtct aaggaagtag gagaggcagt tgcaaagagg tttcatttac
      661 ccggaattag gcaaggatat gggttaacag aaaccacatc tgcaatttta attacacctg
      721 aaggagatga taaaccagga gctgttggga aggttgttcc tttttttgag gctaaagttg
      781 ttgatttaga tactggaaag actttaggag ttaatcaaag aggcgagtta tgtgtaagag
      841 gacctatgat aatgtcagga tacgtaaata atccagaagc tacaaatgca ttgatagata
      901 aagatggttg gttacactca ggcgatatag cttattggga cgaagatgaa cactttttta
      961 tagttgatag attgaaatct ttgattaagt ataaaggtta tcaagtagct ccagcagaat
     1021 tggaaagtat tttattgcaa cacccagctc ttccgtttta gagctagaaa tagcaagtta
     1081 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa
     1141 gcttgggccc gaacaaaaac tcaaagcttg gcactggccg tcgttttaca acgtcgtgac
     1201 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     1261 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     1321 ggcgaatggc gcggccgccc taggttaata cccgagcttc aaggaagatt tcctattaag
     1381 gttgaactta agagcttaag catagacgat ttgaaaaaaa ttttaaaaca aacaaaaaat
     1441 tctttaataa aacaatatgt tgcgatgttt aaggtttatg atttagattt aaagtttagt
     1501 gaggaagcta tagatagaat tgcagagctt acttttaata tgaatcttga gagtgaaaat
     1561 cttggtgcca gaagacttca cggtgttatg gaaatagtgc ttgcagatct tttttttgaa
     1621 gtgcctggca gtaagttgaa aaaatttgaa ataaacttgg actatgttaa taaaaaaata
     1681 caaattaacg aacaaaaaga tttgaattat tatataattt agttaaaagc aattttaaat
     1741 gagggaggtt tccatatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
     1801 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     1861 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     1921 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     1981 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     2041 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     2101 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     2161 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     2221 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     2281 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     2341 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     2401 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     2461 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     2521 gagatcacca aggtagtcgg caaataatga cgtcgccgga gcggtcgagt tctggaccga
     2581 ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga
     2641 cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg
     2701 ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa
     2761 cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga
     2821 gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg
     2881 acacgtgcta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2941 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3001 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3061 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3121 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     3181 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3241 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3301 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3361 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3421 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3481 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3541 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3601 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3661 tcacatgttc tttcctgcac tagatccccg gcaagttcat ccttagggtt ttcatatctc
     3721 ttaaaaagtc aatttctgct tgattaattt cttgtggatt attgtttttg cggtttttca
     3781 ttttttatct ccttgagtat tgaatttttt cttatatata aatatatagc aaaaactatt
     3841 tttgccaact tttttacaaa aaatttttta ttaaaacact tagggctttg ctaaattctc
     3901 ttttaaaaga acttagtaaa gccctatgga tttaagaact gattatactt acgtagtaaa
     3961 aatactacag atattaatag tatagaccat atcaaaatca taatcaattt atatttatct
     4021 aaaattgtag tccatacaaa atacaaaaaa attaggtttt tctaaattct attttaaaag
     4081 aacttagtaa cacctaatct ctaaattact tacaaaattc taaaattaga tttttagttc
     4141 ttcatatttc tttaaaagtt ttttaagaaa atctttttga ttttcaaaaa tttcatgcat
     4201 cataaaacta gtaaatttgg attttttttt gtaaaaatca taactttctt gggttttaag
     4261 ttgaaatctt aatggtttta ttggattttg cctagatttt tttaacgccg gactttcttt
     4321 atcttttata aaattataag aatttttaat accattttct attaaataat cttcttttat
     4381 aagcccctct tgcagtgcag ttgctaatct taaatactta tacgcttgag tttttgctac
     4441 atcaaagtct tttataaaat caagaaaagt tttataacca tcaaatttat aatattgatt
     4501 atcttttatt tcttttaaaa ttttaatatt ttccaccctg tggaatattt cttttttgaa
     4561 attgtcattt aacttttcct ttagtgtctt gtaataagaa attttacttt cattaatcat
     4621 taaattttgt tcaacataag tatctatctt ttttgttaaa actttaacaa cacgtttttt
     4681 ttccataaaa attttccttt atttttgcaa cattttcctt aattgatata attgttacta
     4741 ttttagattt tttaatatcc acttaagaat taatccctca ctagaatatt ttttattata
     4801 gaattcatat acatgttttt ctaaatctat aaatttatca acaattgctt tattataggg
     4861 tgtatctatc ttttcttttc ttaataatcg tctcatattc tttaaataac aacaaacact
     4921 gccggtttta aacctaaatt ctatgtaata tatttttgaa aaagtatata cattttcaac
     4981 cccgtttatt tcatacttta caaaaacatt atttattggt tttctataac catagtaaat
     5041 gcctaaaaat ttgtccccgt ctttcatagg atatagataa agtttttgaa tctctgattt
     5101 atctaataat ttttcaaaaa aaattataca ccgcttttgt ttgttactaa ctactctatg
     5161 caatgccttt aatatttttg tgtgatatat atttttatca cctttttttt ctttcagcag
     5221 gaagatactt tcttttggaa atttaatttt ttgttcattt attcttgcag ccttaagtcc
     5281 gattttcata tttaatcctt atatagcctt tctatttaat tcttttaaac tcaataaact
     5341 tttttggttt ttcattagtt ctaataattc gcaataatat gtgcaaaaca ctttattgta
     5401 ttctattttc ttttggttat tcaaatattc ttttataatt ggctttagaa gttcaatatt
     5461 tacttctttt tttagttgtt caataaggat attgaaaatg tttgttttta aatcttgata
     5521 attttgttga gaattttctt tttttctttc aattgacttc tctaatttac gctttatatt
     5581 gtttaaatcg ctatatttat gactttcaat aataaaatga ggcttaaatt tgtaattttc
     5641 gtatattttt tggaaacttg tttctaattg tttagaatta tattcatttt tttcaaattc
     5701 tttttgagtg ttgcatagaa tttcctttaa tttttcttgt ttttctttca tacaagattt
     5761 agtaaaatgt atatttttat tttttagtaa tttaatttct attattttta tgattttaag
     5821 tattttgatt atattatctt tatcaatatt taaattgaat aaaattgtaa gaaaaattga
     5881 aagaattttt ttacataaaa agttacattt attgaagtaa tttcttactt gatacttttc
     5941 tatttcatta atctttcttt cttcttttat attattttta ttatttaaac actcctttaa
     6001 atctacactc ccatttttag taaatctatc tttaaggccc cttgaaactc tattttgaaa
     6061 tcttaaatct tttttttctt gaaagtattt atagattttt tggtaacatt ctttttttga
     6121 aaaattaagt tgatagtata tttcggtccc caaatttacc cctaagtgtt tgtgataatt
     6181 agttgtgact ttaaattctt tttctaattt gtaaaggtaa ttttgcagtg tttttagttt
     6241 aacgggtgtt tgactatttc ttttaaggtt ttcattaaag taatagagta tgtttttttg
     6301 tgtgtatttc ttatatgttt tatttatgta cgctagtgtt gagataagga ctactaattt
     6361 atactgtaat ttattgtagc aagttgtggt tttactggtt ttgtttatat tagataagtt
     6421 ttccatttat agctccttga ataccggatt ttttatctat aaagtatata tatcacaatt
     6481 ttgatttaaa agtaaattct ttttttaaaa atacaaaaat tttttataaa ttatatagac
     6541 catgatattg tattaatact aattgatttt aatttaaggt tgaactaaaa taaacatagt
     6601 tttgtagaaa ataatttttc attattttgc tacttgatat tggatcgtaa aaatattagg
     6661 gctttacaaa gttcttttaa aagagaattt agtaaagccc taagtctttt aataaaaaat
     6721 tttttgtaaa a
//
