LOCUS       sequence-269481-        5505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for constitutive expression of a single guide RNA from Streptococcus
            pyogenes with a seed region that targets ctrA gene's promoter from 
            Caulobacter crescentus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5505)
  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 5505)
  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(Spy)_ctrA
FEATURES             Location/Qualifiers
     source          1..5505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..76
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      82..125
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(268..284)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(268..284)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(316..335)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(317..335)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(345..362)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(345..361)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(354..376)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1070..1732)
                     /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      1733..2504
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             3984..4778
                     /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(4038..4057)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4648..4667
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5126..5172
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt gataaaacga aaggcccagt ctttcgactg agcctttcgt
      121 tttataaacg ggtcttgagg ggttttttaa gcttctgcag ctggatccat ggatatctag
      181 atttccgcaa aataaaaaag gggacctcta gggtccccaa ttaattagct aaagccgcta
      241 gcccatggga attcctgcag cccgggggat ccactagttc tagagcggcc gccaccgcgg
      301 tggagctcca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
      361 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      421 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      481 cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
      541 atttttgtta aatcagctca ttttttaacc aataggccgt actgcgatga gtggcagggc
      601 ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggttg ctacgcctga
      661 ataagtgata ataagcggat gaatggcaga aattcgaaag caaattcgac ccggtcgtcg
      721 gttcagggca gggtcgttaa atagccgctt atgtctattg ctggtttacc ggtttattga
      781 ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg aggccagttt gctcaggctc
      841 tccccgtgga ggtaataatt gacgatatga tcatttattc tgcctcccag agcctgataa
      901 aaacggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
      961 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcga ggcggctaca
     1021 gccgatagtc tggaacagcg cacttacggg ttgctgcgca acccaagtgc taccggcgcg
     1081 gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc gtccagaaaa cacggctcat
     1141 cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc ctccgtttcg gtcaaggctg
     1201 gcaggtctgg ttccatgccc ggaatgccgg gctggctggg cggctcctcg ccggggccgg
     1261 tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg gcgcaggtcg ccatgcccca
     1321 acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc ggcactgaac accgacaggc
     1381 gcaactggtc gcggggctgg ccccacgcca cgcggtcatt gaccacgtag gccgacacgg
     1441 tgccggggcc gttgagcttc acgacggaga tccagcgctc ggccaccaag tccttgactg
     1501 cgtattggac cgtccgcaaa gaacgtccga tgagcttgga aagtgttttc tggctgacca
     1561 ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg cagcagcatt gccgccgtgg
     1621 gtttcctcgc aataagcccg gcccacgcct catgcgcttt gcgttccgtt tgcacccagt
     1681 gaccgggctt gttcttggct tgaatgccga tttctctgga ctgcgtggcc atgcttatct
     1741 ccatgcggta ggggtgccgc acggttgcgg caccatgcgc aatcagctgc aacttttcgg
     1801 cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc aattacagat tttctttaac
     1861 ctacgcaatg agctattgcg gggggtgccg caatgagctg ttgcgtaccc ccctttttta
     1921 agttgttgat ttttaagtct ttcgcatttc gccctatatc tagttctttg gtgcccaaag
     1981 aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg ctccccctcc ggcaaaaagt
     2041 ggcccctccg gggcttgttg atcgactgcg cggccttcgg ccttgcccaa ggtggcgctg
     2101 cccccttgga acccccgcac tcgccgccgt gaggctcggg gggcaggcgg gcgggcttcg
     2161 cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca aggccaagcc
     2221 gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa ccggcaagcg
     2281 aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa attgatgggg
     2341 caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct gggtagccgg
     2401 tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag cttgtccagc
     2461 agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg ccatcgtcca
     2521 catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc ggagagcaag
     2581 cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa gtctagtgag
     2641 tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc cgtcgagccg
     2701 gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg atgcaagaag
     2761 ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga gcgcgagacg
     2821 cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag cagcaccgat
     2881 gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga cgctgtgttg
     2941 gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag ccaagaacag
     3001 caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg ggcggatcgc
     3061 atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc gttcgtggtg
     3121 ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa agcgcagatg
     3181 acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca acggggcatc
     3241 gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct ggagcggcca
     3301 ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta tgcaccgcag
     3361 ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc cgtggccgac
     3421 cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc cgcaggagcg
     3481 cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct tcgggagcgc
     3541 ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc caaggccgcc
     3601 gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt ccagcgccag
     3661 aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa gtgccacctg
     3721 ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc
     3781 aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa
     3841 gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa
     3901 actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag
     3961 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     4021 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4081 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4141 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4201 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4261 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4321 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4381 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4441 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4501 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4561 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4621 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4681 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4741 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4801 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4861 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4921 ggatctcatg ctggagttct tcgcccaccc ccatgggcaa atattatacg caaggcgaca
     4981 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     5041 gcagaatgct taatgaatta caacagtttt tatataagct ttcagtcggc gcgaatgctc
     5101 cacgccgcgc ccagcggcgc ggatccaaat aaaacgaaag gctcagtcga aagactgggc
     5161 ctttcgtttt atctgttgtt tgtcgctgca gccagccgtg gtcgggcagc aggtagaagg
     5221 cgccctcgtc ctgatcctcg cccgaaacct ccagcccccg gtcgatggct tcgacgacat
     5281 agccggccgc gcggcaggtg tcggtgagcg cggccagcag ggcggcttcc tggtcagggg
     5341 tcaggtcggt catgggcaag aggtccaggt cgtggtttgt cggcggcttc tagcatggac
     5401 cgcccgcgcc cgtgaggccg aggatttcgc gctggtcaga caacctactt gccgtcccca
     5461 catgttagcg ctaccaagtg ccgacgttaa tttaagactg gttaa
//
