LOCUS       sequence-269542-        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 non-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)_gcrA1
FEATURES             Location/Qualifiers
     source          1..5515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1..47
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      472..515
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(658..674)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(658..674)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(706..725)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(707..725)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(735..752)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(735..751)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(744..766)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1460..2122)
                     /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      2123..2894
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4374..5168
                     /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(4428..4447)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5038..5057
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
       61 ctgcagccag ccgtggtcgg gcagcaggta gaaggcgccc tcgtcctgat cctcgcccga
      121 aacctccagc ccccggtcga tggcttcgac gacatagccg gccgcgcggc aggtgtcggt
      181 gagcgcggcc agcagggcgg cttcctggtc aggggtcagg tcggtcatgg gcaagaggtc
      241 caggtcgtgg tttgtcggcg gcttctagca tggaccgccc gcgcccgtga ggccgaggat
      301 ttcgcgctgg tcagacaacc tacttgccgt ccccacatgt tagcgctacc aagtgccgac
      361 acgcccgtca ccctatatag ttttagagct gtgaaaacag cgagttaaaa taaggcttag
      421 tccgtactca acttgaaaag gtggcaccga ttcggtgttt tttttttttt gataaaacga
      481 aaggcccagt ctttcgactg agcctttcgt tttataaacg ggtcttgagg ggttttttaa
      541 gcttctgcag ctggatccat ggatatctag atttccgcaa aataaaaaag gggacctcta
      601 gggtccccaa ttaattagct aaagccgcta gcccatggga attcctgcag cccgggggat
      661 ccactagttc tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg
      721 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
      781 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
      841 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa
      901 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
      961 aataggccgt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg
     1021 tgcccttaaa cgcctggttg ctacgcctga ataagtgata ataagcggat gaatggcaga
     1081 aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt
     1141 atgtctattg ctggtttacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct
     1201 caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga
     1261 tcatttattc tgcctcccag agcctgataa aaacggtgaa tccgttagcg aggtgccgcc
     1321 ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca
     1381 gacaaggtat agggcggcga ggcggctaca gccgatagtc tggaacagcg cacttacggg
     1441 ttgctgcgca acccaagtgc taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg
     1501 gctcgccatc gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc
     1561 gttcccattc ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg
     1621 gctggctggg cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg
     1681 tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa
     1741 ccaccacggc ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca
     1801 cgcggtcatt gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga
     1861 tccagcgctc ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga
     1921 tgagcttgga aagtgtcttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca
     1981 cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct
     2041 catgcgcttt gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga
     2101 tttctctgga ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg
     2161 caccatgcgc aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa
     2221 agtggcagtc aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg
     2281 caatgagctg ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc
     2341 gccctatatc tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc
     2401 ttcggcgcgg ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg
     2461 cggccttcgg ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt
     2521 gaggctcggg gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt
     2581 gggtcgttcc aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc
     2641 gccttccact tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggatgctttt
     2701 ccccagagaa aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg
     2761 tgggtatgtg gtcgaaggct gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc
     2821 aggcgcagcc tgtccatcag cttgtccagc agggttgtcc acgtgccgag cgaagcgagc
     2881 cagccggtgg ccgctcgcgg ccatcgtcca catatccacg ggctggcaag ggagcgcagc
     2941 gaccgcgcag ggcgaagccc ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg
     3001 tcatgacttt gcgaagcaaa gtctagtgag tatactcaag cattgagtgg cccgccggag
     3061 gcaccgcctt gcgctgcccc cgtcgagccg gttggacacc aaaagggagg ggcaggcatg
     3121 gcggcatacg cgatcatgcg atgcaagaag ctggcgaaaa tgtgcaacgt ggcggccagt
     3181 ctcaagcacg cctaccgcga gcgcgagacg cccaacgctg acgccagcag gacgccagag
     3241 aacgagcact gggcggccag cagcaccgat gaagcgatgg gccgactgcg cgagttgctg
     3301 ccagagaagc ggcgcaagga cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg
     3361 gaatggtgga agtcggccag ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag
     3421 tggctggcgg acaagtacgg ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa
     3481 acaagcccgc acatgaccgc gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc
     3541 aaggagttca tcggcaacaa agcgcagatg acccgcgacc agaccacgtt tgcggccgct
     3601 gtggccgatc tagggctgca acggggcatc gagggcagca aggcacgtca cacgcgcatt
     3661 caggcgttct acgaggccct ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa
     3721 gcggtcgagc cacgcgccta tgcaccgcag ggattggccg aaaagctggg aatctcaaag
     3781 cgcgttgaga cgccggaagc cgtggccgac cggctgacaa aagcggttcg gcaggggtat
     3841 gagcctgccc tacaggccgc cacaggagcg cgtgagatgc gcaagaaggc cgatcaagcc
     3901 caagagacgg cccgagacct tcgggagcgc ctgaagcccg ttctggacgc cctggggccg
     3961 ttgaatcggg atatgcaggc caaggccgcc gcgatcatca aggccgtggg cgaaaagctg
     4021 ctgacggaac agcgggaagt ccagcgccag aaacaggccc agcgccagca ggaacgcggg
     4081 cgcgcacatt tccccgaaaa gtgccacctg ggatgaatgt cagctactgg gctatctgga
     4141 caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat
     4201 agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct
     4261 ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct
     4321 gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     4381 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     4441 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     4501 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     4561 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcaact gtgctcgacg
     4621 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     4681 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4741 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4801 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4861 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4921 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4981 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     5041 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     5101 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     5161 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     5221 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     5281 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     5341 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     5401 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtttt
     5461 tatataagct ttcagtcggc gcgaatgctc cacgccgcgc ccagcggcgc ggatc
//
