LOCUS       sequence-267423-        5510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for constitutive expression of a single guide RNA from Streptococcus
            pasteurianus with a seed region that targets gcrA gene's promoter 
            from Caulobacter crescentus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5510)
  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 5510)
  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(Spa)_gcrA3
FEATURES             Location/Qualifiers
     source          1..5510
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2087..2881
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2141..2160)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2751..2770
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      3229..3275
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3695..3738
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3881..3897)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3881..3897)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3929..3948)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3930..3948)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3958..3975)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3958..3974)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3967..3989)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(4683..5345)
                     /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      join(5346..5510,1..607)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 ctgttgcgta cccccctttt ttaagttgtt gatttttaag tctttcgcat ttcgccctat
       61 atctagttct ttggtgccca aagaagggca cccctgcggg gttcccccac gccttcggcg
      121 cggctccccc tccggcaaaa agtggcccct ccggggcttg ttgatcgact gcgcggcctt
      181 cggccttgcc caaggtggcg ctgccccctt ggaacccccg cactcgccgc cgtgaggctc
      241 ggggggcagg cgggcgggct tcgcccttcg actgccccca ctcgcatagg cttgggtcgt
      301 tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg cgagccttga cccgccttcc
      361 acttggtgtc caaccggcaa gcgaagcgcg caggccgcag gccggaggct tttccccaga
      421 gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg cagttggagc cggtgggtat
      481 gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt caagctcgtg ggcaggcgca
      541 gcctgtccat cagcttgtcc agcagggttg tccacgggcc gagcgaagcg agccagccgg
      601 tggccgctcg cggccatcgt ccacatatcc acgggctggc aagggagcgc agcgaccgcg
      661 cagggcgaag cccggagagc aagcccgtag ggcgccgcag ccgccgtagg cggtcacgac
      721 tttgcgaagc aaagtctagt gagtatactc aagcattgag tggcccgccg gaggcaccgc
      781 cttgcgctgc ccccgtcgag ccggttggac accaaaaggg aggggcaggc atggcggcat
      841 acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc agtctcaagc
      901 acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca gagaacgagc
      961 actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg ctgccagaga
     1021 agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc ccggaatggt
     1081 ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac aagtggctgg
     1141 cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac gaaaccagcc
     1201 cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg gccaaggagt
     1261 tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc gctgtggccg
     1321 atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc attcaggcgt
     1381 tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg caagcggtcg
     1441 agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca aagcgcgttg
     1501 agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg tatgagcctg
     1561 ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa gcccaagaga
     1621 cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg ccgttgaatc
     1681 gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag ctgctgacgg
     1741 aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc gggcgcgcac
     1801 atttccccga aaagtgccac ctgggatgaa tgtcagctac tgggctatct ggacaaggga
     1861 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga
     1921 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa
     1981 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg
     2041 caggggatca agatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     2101 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     2161 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     2221 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
     2281 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     2341 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     2401 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     2461 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     2521 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     2581 cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt
     2641 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     2701 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     2761 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     2821 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     2881 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     2941 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3001 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca cccccatggg
     3061 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     3121 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt ttttatataa
     3181 gctttcagtc ggcgcgaatg ctccacgccg cgcccagcgg cgcggatcca aataaaacga
     3241 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcgct gcagccagcc
     3301 gtggtcgggc agcaggtaga aggcgccctc gtcctgatcc tcgcccgaaa cctccagccc
     3361 ccggtcgatg gcttcgacga catagccggc cgcgcggcag gtgtcggtga gcgcggccag
     3421 cagggcggct tcctggtcag gggtcaggtc ggtcatgggc aagaggtcca ggtcgtggtt
     3481 tgtcggcggc ttctagcatg gaccgcccgc gcccgtgagg ccgaggattt cgcgctggtc
     3541 agacaaccta cttgccgtcc ccacatgtta gcgctaccaa gtgccgaccg gatcgatcac
     3601 catatatagt ttttgtactc gaaagagcct acaaagataa ggctttatgc cgaattcaag
     3661 caccccatgt tttgacatga ggtgcttttt tttgataaaa cgaaaggccc agtctttcga
     3721 ctgagccttt cgttttataa acgggtcttg aggggttttt taagcttctg cagctggatc
     3781 catggatatc tagatttccg caaaataaaa aaggggacct ctagggtccc caattaatta
     3841 gctaaagccg ctagcccatg ggaattcctg cagcccgggg gatccactag ttctagagcg
     3901 gccgccaccg cggtggagct ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     3961 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     4021 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     4081 ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta
     4141 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgtactgcga
     4201 tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg
     4261 ttgctacgcc tgaataagtg ataataagcg gatgaatggc agaaattcga aagcaaattc
     4321 gacccggtcg tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggttt
     4381 accggtttat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag
     4441 tttgctcagg ctctccccgt ggaggtaata attgacgata tgatcattta ttctgcctcc
     4501 cagagcctga taaaaacggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
     4561 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     4621 cgaggcggct acagccgata gtctggaaca gcgcacttac gggttgctgc gcaacccaag
     4681 tgctaccggc gcggcagcgt gacccgtgtc ggcggctcca acggctcgcc atcgtccaga
     4741 aaacacggct catcgggcat cggcaggcgc tgctgcccgc gccgttccca ttcctccgtt
     4801 tcggtcaagg ctggcaggtc tggttccatg cccggaatgc cgggctggct gggcggctcc
     4861 tcgccggggc cggtcggtag ttgctgctcg cccggataca gggtcgggat gcggcgcagg
     4921 tcgccatgcc ccaacagcga ttcgtcctgg tcgtcgtgat caaccaccac ggcggcactg
     4981 aacaccgaca ggcgcaactg gtcgcggggc tggccccacg ccacgcggtc attgaccacg
     5041 taggccgaca cggtgccggg gccgttgagc ttcacgacgg agatccagcg ctcggccacc
     5101 aagtccttga ctgcgtattg gaccgtccgc aaagaacgtc cgatgagctt ggaaagtgtt
     5161 ttctggctga ccaccacggc gttctggtgg cccatctgcg ccacgaggtg atgcagcagc
     5221 attgccgccg tgggtttcct cgcaataagc ccggcccacg cctcatgcgc tttgcgttcc
     5281 gtttgcaccc agtgaccggg cttgttcttg gcttgaatgc cgatttctct ggactgcgtg
     5341 gccatgctta tctccatgcg gtaggggtgc cgcacggttg cggcaccatg cgcaatcagc
     5401 tgcaactttt cggcagcgcg acaacaatta tgcgttgcgt aaaagtggca gtcaattaca
     5461 gattttcttt aacctacgca atgagctatt gcggggggtg ccgcaatgag
//
