LOCUS       sequence-308060-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  flaB gRNA expression in B. burgdorferi.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  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 6540)
  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: pBSV2_Psyn-sgRNAflaB
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1251..1272
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1287..1292,1293..1310,1311..1317)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1325..1341
                     /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     1330..1352
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1349..1365
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1349..1365
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1402..1436
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1458..1533
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1576..1593)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1576..1592)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1585..1607)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2147..2956
                     /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(2214..2233)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     regulatory      3207..3216
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3213..3587
                     /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      3725..4313
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4214..4233
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatctttatc aatatttaaa ttgaataaaa ttgtaagaaa aattgaaaga atttttttac
       61 ataaaaagtt acatttattg aagtaatttc ttacttgata cttttctatt tcattaatct
      121 ttctttcttc ttttatatta tttttattat ttaaacactc ctttaaatct acactcccat
      181 ttttagtaaa tctatcttta aggccccttg aaactctatt ttgaaatctt aaatcttttt
      241 tttcttgaaa gtatttatag attttttggt aacattcttt ttttgaaaaa ttaagttgat
      301 agtatatttc ggtccccaaa tttaccccta agtgtttgtg ataattagtt gtgactttaa
      361 attctttttc taatttgtaa aggtaatttt gcagtgtttt tagtttaacg ggtgtttgac
      421 tatttctttt aaggttttca ttaaagtaat agagtatgtt tttttgtgtg tatttcttat
      481 atgttttatt tatgtacgct agtgttgaga taaggactac taatttatac tgtaatttat
      541 tgtagcaagt tgtggtttta ctggttttgt ttatattaga taagttttcc atttatagct
      601 ccttgaatac cggatttttt atctataaag tatatatatc acaattttga tttaaaagta
      661 aattcttttt ttaaaaatac aaaaattttt tataaattat atagaccatg atattgtatt
      721 aatactaatt gattttaatt taaggttgaa ctaaaataaa catagttttg tagaaaataa
      781 tttttcatta ttttgctact tgatattgga tcgtaaaaat attagggctt tacaaagttc
      841 ttttaaaaga gaatttagta aagccctaag tcttttaata aaaaattttt tgtaaaaaag
      901 ttggcaaaaa tagtttttgc tatatattta tatataagaa aaaattcaat actcaaggag
      961 ataaaaaatg gaaaacttaa acaataatcc acaaaaaatt aatcaaataa ataagcaaac
     1021 taattttatt ggttttgaaa attttataga acttgataat caagttaatc aaactaaaga
     1081 aaaaagtaaa ttaagtaaga taggtaagaa attacccgga ataagcagtc aagtctagtg
     1141 cggccgcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     1201 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1261 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1321 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgaattc
     1381 gagctcgaat tctaaagatc tttgacagct agctcagtcc taggtataat actagtgaaa
     1441 atttaaattt ctgacttgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1501 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttggg cccgaacaaa
     1561 aactcaaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     1621 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     1681 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcggccgct
     1741 acccgagctt caaggaagat ttcctattaa ggttgaactt aagagcttaa gcatagacga
     1801 tttgaaaaaa attttaaaac aaacaaaaaa ttctttaata aaacaatatg ttgcgatgtt
     1861 taaggtttat gatttagatt taaagtttag tgaggaagct atagatagaa ttgcagagct
     1921 tacttttaat atgaatcttg agagtgaaaa tcttggtgcc agaagacttc acggtgttat
     1981 ggaaatagtg cttgcagatc ttttttttga agtgcctggc agtaagttga aaaaatttga
     2041 aataaacttg gactatgtta ataaaaaaat acaaattaac gaacaaaaag atttgaatta
     2101 ttatataatt tagttaaaag caattttaaa tgagggaggt ttccatatga gccatattca
     2161 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     2221 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     2281 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     2341 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     2401 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     2461 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     2521 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     2581 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     2641 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     2701 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     2761 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     2821 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     2881 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     2941 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     3001 gacttgacgg gacggcgctt acaatttcct gatgcggtat tttctcctta cgcatctgtg
     3061 cggtatttca caccgcatca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3121 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3181 tgcttcaata atagcacgtg aggagggcca ccatggccaa gttgaccagt gccgttccgg
     3241 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     3301 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     3361 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     3421 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     3481 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     3541 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctaaaac
     3601 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3661 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3721 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3781 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3841 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     3901 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3961 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4021 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     4081 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     4141 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4201 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4261 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4321 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4381 ctgcactaga tccccggcaa gttcatcctt agggttttca tatctcttaa aaagtcaatt
     4441 tctgcttgat taatttcttg tggattattg tttttgcggt ttttcatttt ttatctcctt
     4501 gagtattgaa ttttttctta tatataaata tatagcaaaa actatttttg ccaacttttt
     4561 tacaaaaaat tttttattaa aacacttagg gctttgctaa attctctttt aaaagaactt
     4621 agtaaagccc tatggattta agaactgatt atacttacgt agtaaaaata ctacagatat
     4681 taatagtata gaccatatca aaatcataat caatttatat ttatctaaaa ttgtagtcca
     4741 tacaaaatac aaaaaaatta ggtttttcta aattctattt taaaagaact tagtaacacc
     4801 taatctctaa attacttaca aaattctaaa attagatttt tagttcttca tatttcttta
     4861 aaagtttttt aagaaaatct ttttgatttt caaaaatttc atgcatcata aaactagtaa
     4921 atttggattt ttttttgtaa aaatcataac tttcttgggt tttaagttga aatcttaatg
     4981 gttttattgg attttgccta gattttttta acgccggact ttctttatct tttataaaat
     5041 tataagaatt tttaatacca ttttctatta aataatcttc ttttataagc ccctcttgca
     5101 gtgcagttgc taatcttaaa tacttatacg cttgagtttt tgctacatca aagtctttta
     5161 taaaatcaag aaaagtttta taaccatcaa atttataata ttgattatct tttatttctt
     5221 ttaaaatttt aatattttcc accctgtgga atatttcttt tttgaaattg tcatttaact
     5281 tttcctttag tgtcttgtaa taagaaattt tactttcatt aatcattaaa ttttgttcaa
     5341 cataagtatc tatctttttt gttaaaactt taacaacacg ttttttttcc ataaaaattt
     5401 tcctttattt ttgcaacatt ttccttaatt gatataattg ttactatttt agatttttta
     5461 atatccactt aagaattaat ccctcactag aatatttttt attatagaat tcatatacat
     5521 gtttttctaa atctataaat ttatcaacaa ttgctttatt atagggtgta tctatctttt
     5581 cttttcttaa taatcgtctc atattcttta aataacaaca aacactgccg gttttaaacc
     5641 taaattctat gtaatatatt tttgaaaaag tatatacatt ttcaaccccg tttatttcat
     5701 actttacaaa aacattattt attggttttc tataaccata gtaaatgcct aaaaatttgt
     5761 ccccgtcttt cataggatat agataaagtt tttgaatctc tgatttatct aataattttt
     5821 caaaaaaaat tatacaccgc ttttgtttgt tactaactac tctatgcaat gcctttaata
     5881 tttttgtgtg atatatattt ttatcacctt ttttttcttt cagcaggaag atactttctt
     5941 ttggaaattt aattttttgt tcatttattc ttgcagcctt aagtccgatt ttcatattta
     6001 atccttatat agcctttcta tttaattctt ttaaactcaa taaacttttt tggtttttca
     6061 ttagttctaa taattcgcaa taatatgtgc aaaacacttt attgtattct attttctttt
     6121 ggttattcaa atattctttt ataattggct ttagaagttc aatatttact tcttttttta
     6181 gttgttcaat aaggatattg aaaatgtttg tttttaaatc ttgataattt tgttgagaat
     6241 tttctttttt tctttcaatt gacttctcta atttacgctt tatattgttt aaatcgctat
     6301 atttatgact ttcaataata aaatgaggct taaatttgta attttcgtat attttttgga
     6361 aacttgtttc taattgttta gaattatatt catttttttc aaattctttt tgagtgttgc
     6421 atagaatttc ctttaatttt tcttgttttt ctttcataca agatttagta aaatgtatat
     6481 ttttattttt tagtaattta atttctatta tttttatgat tttaagtatt ttgattatat
//
