LOCUS       sequence-266829-        5515 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for constitutive expression of a single guide RNA from Streptococcus
            thermophilus #3 with a seed region that targets gcrA gene's promoter
            on the template strand in Caulobacter crescentus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5515)
  AUTHORS   Guzzo M, Castro LK, Reisch CR, Guo MS, Laub MT
  TITLE     A CRISPR Interference System for Efficient and Rapid Gene Knockdown 
            in Caulobacter crescentus.
  JOURNAL   mBio. 2020 Jan 14;11(1):e02415-19. doi: 10.1128/mBio.02415-19.
  PUBMED    31937638
REFERENCE   2  (bases 1 to 5515)
  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"; doi: "10.1128/mBio"; 
            journalName: "mBio"; date: "2020-01-14- 14"; volume: "11"; issue: 
            "1"; pages: "e02415-19"
COMMENT     Sequence Label: pBXMCS-2 Pconstitutive-sgRNA(Sth3)_gcrA2
FEATURES             Location/Qualifiers
     source          1..5515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      385..428
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(571..587)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(571..587)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(619..638)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(620..638)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(648..665)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(648..664)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(657..679)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1373..2035)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      2036..2807
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4287..5081
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCATVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4341..4360)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4951..4970
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5429..5475
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gtagaaggcg ccctcgtcct gatcctcgcc cgaaacctcc agcccccggt cgatggcttc
       61 gacgacatag ccggccgcgc ggcaggtgtc ggtgagcgcg gccagcaggg cggcttcctg
      121 gtcaggggtc aggtcggtca tgggcaagag gtccaggtcg tggtttgtcg gcggcttcta
      181 gcatggaccg cccgcgcccg tgaggccgag gatttcgcgc tggtcagaca acctacttgc
      241 cgtccccaca tgttagcgct accaagtgcc gacggatcga tcaccatata tagttttaga
      301 gctgtgaaaa cagcgagtta aaataaggct tagtccgtac tcaacttgaa aaggtggcac
      361 cgattcggtg tttttttttt tttgataaaa cgaaaggccc agtctttcga ctgagccttt
      421 cgttttataa acgggtcttg aggggttttt taagcttctg cagctggatc catggatatc
      481 tagatttccg caaaataaaa aaggggacct ctagggtccc caattaatta gctaaagccg
      541 ctagcccatg ggaattcctg cagcccgggg gatccactag ttctagagcg gccgccaccg
      601 cggtggagct ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
      661 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
      721 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
      781 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
      841 taaatttttg ttaaatcagc tcatttttta accaataggc cgtactgcga tgagtggcag
      901 ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc
      961 tgaataagtg ataataagcg gatgaatggc agaaattcga aagcaaattc gacccggtcg
     1021 tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat
     1081 tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag tttgctcagg
     1141 ctctccccgt ggaggtaata attgacgata tgatcattta ttctgcctcc cagagcctga
     1201 taaaaacggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg
     1261 gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg cgaggcggct
     1321 acagccgata gtctggaaca gcgcacttac gggttgctgc gcaacccaag tgctaccggc
     1381 gcggcagcgt gacccgtgtc ggcggctcca acggctcgcc atcgtccaga aaacacggct
     1441 catcgggcat cggcaggcgc tgctgcccgc gccgttccca ttcctccgtt tcggtcaagg
     1501 ctggcaggtc tggttccatg cccggaatgc cgggctggct gggcggctcc tcgccggggc
     1561 cggtcggtag ttgctgctcg cccggataca gggtcgggat gcggcgcagg tcgccatgcc
     1621 ccaacagcga ttcgtcctgg tcgtcgtgat caaccaccac ggcggcactg aacaccgaca
     1681 ggcgcaactg gtcgcggggc tggccccacg ccacgcggtc attgaccacg taggccgaca
     1741 cggtgccggg gccgttgagc ttcacgacgg agatccagcg ctcggccacc aagtccttga
     1801 ctgcgtattg gaccgtccgc aaagaacgtc cgatgagctt ggaaagtgtc ttctggctga
     1861 ccaccacggc gttctggtgg cccatctgcg ccacgaggtg atgcagcagc attgccgccg
     1921 tgggtttcct cgcaataagc ccggcccacg cctcatgcgc tttgcgttcc gtttgcaccc
     1981 agtgaccggg cttgttcttg gcttgaatgc cgatttctct ggactgcgtg gccatgctta
     2041 tctccatgcg gtaggggtgc cgcacggttg cggcaccatg cgcaatcagc tgcaactttt
     2101 cggcagcgcg acaacaatta tgcgttgcgt aaaagtggca gtcaattaca gattttcttt
     2161 aacctacgca atgagctatt gcggggggtg ccgcaatgag ctgttgcgta cccccctttt
     2221 ttaagttgtt gatttttaag tctttcgcat ttcgccctat atctagttct ttggtgccca
     2281 aagaagggca cccctgcggg gttcccccac gccttcggcg cggctccccc tccggcaaaa
     2341 agtggcccct ccggggcttg ttgatcgact gcgcggcctt cggccttgcc caaggtggcg
     2401 ctgccccctt ggaacccccg cactcgccgc cgtgaggctc ggggggcagg cgggcgggct
     2461 tcgcccttcg actgccccca ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa
     2521 gccgctgcgc ggtcgctgcg cgagccttga cccgccttcc acttggtgtc caaccggcaa
     2581 gcgaagcgcg caggccgcag gccggatgct tttccccaga gaaaattaaa aaaattgatg
     2641 gggcaaggcc gcaggccgcg cagttggagc cggtgggtat gtggtcgaag gctgggtagc
     2701 cggtgggcaa tccctgtggt caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc
     2761 agcagggttg tccacgtgcc gagcgaagcg agccagccgg tggccgctcg cggccatcgt
     2821 ccacatatcc acgggctggc aagggagcgc agcgaccgcg cagggcgaag cccggagagc
     2881 aagcccgtag ggcgccgcag ccgccgtagg cggtcatgac tttgcgaagc aaagtctagt
     2941 gagtatactc aagcattgag tggcccgccg gaggcaccgc cttgcgctgc ccccgtcgag
     3001 ccggttggac accaaaaggg aggggcaggc atggcggcat acgcgatcat gcgatgcaag
     3061 aagctggcga aaatgtgcaa cgtggcggcc agtctcaagc acgcctaccg cgagcgcgag
     3121 acgcccaacg ctgacgccag caggacgcca gagaacgagc actgggcggc cagcagcacc
     3181 gatgaagcga tgggccgact gcgcgagttg ctgccagaga agcggcgcaa ggacgctgtg
     3241 ttggcggtcg agtacgtcat gacggccagc ccggaatggt ggaagtcggc cagccaagaa
     3301 cagcaggcgg cgttcttcga gaaggcgcac aagtggctgg cggacaagta cggggcggat
     3361 cgcatcgtga cggccagcat ccaccgtgac gaaacaagcc cgcacatgac cgcgttcgtg
     3421 gtgccgctga cgcaggacgg caggctgtcg gccaaggagt tcatcggcaa caaagcgcag
     3481 atgacccgcg accagaccac gtttgcggcc gctgtggccg atctagggct gcaacggggc
     3541 atcgagggca gcaaggcacg tcacacgcgc attcaggcgt tctacgaggc cctggagcgg
     3601 ccaccagtgg gccacgtcac catcagcccg caagcggtcg agccacgcgc ctatgcaccg
     3661 cagggattgg ccgaaaagct gggaatctca aagcgcgttg agacgccgga agccgtggcc
     3721 gaccggctga caaaagcggt tcggcagggg tatgagcctg ccctacaggc cgccacagga
     3781 gcgcgtgaga tgcgcaagaa ggccgatcaa gcccaagaga cggcccgaga ccttcgggag
     3841 cgcctgaagc ccgttctgga cgccctgggg ccgttgaatc gggatatgca ggccaaggcc
     3901 gccgcgatca tcaaggccgt gggcgaaaag ctgctgacgg aacagcggga agtccagcgc
     3961 cagaaacagg cccagcgcca gcaggaacgc gggcgcgcac atttccccga aaagtgccac
     4021 ctgggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     4081 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     4141 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     4201 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     4261 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     4321 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4381 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4441 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     4501 cgacgggcgt tccttgcgca actgtgctcg acgttgtcac tgaagcggga agggactggc
     4561 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     4621 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4681 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4741 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4801 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4861 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4921 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4981 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5041 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5101 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     5161 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     5221 cggggatctc atgctggagt tcttcgccca cccccatggg caaatattat acgcaaggcg
     5281 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     5341 tcggcagaat gcttaatgaa ttacaacagt ttttatataa gctttcagtc ggcgcgaatg
     5401 ctccacgccg cgcccagcgg cgcggatcca aataaaacga aaggctcagt cgaaagactg
     5461 ggcctttcgt tttatctgtt gtttgtcgct gcagccagcc gtggtcgggc agcag
//
