LOCUS       sequence-258095-        5557 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 5557)
  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 5557)
  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..5557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        201..276
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      282..325
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(468..484)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(468..484)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(516..535)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(517..535)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(545..562)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(545..561)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(554..576)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1270..1932)
                     /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      1933..2704
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4184..4978
                     /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(4238..4257)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4848..4867
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5326..5372
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gagcgcggcc agcagggcgg cttcctggtc aggggtcagg tcggtcatgg gcaagaggtc
       61 caggtcgtgg tttgtcggcg gcttctagca tggaccgccc gcgcccgtga ggccgaggat
      121 ttcgcgctgg tcagacaacc tacttgccgt ccccacatgt tagcgctacc aagtgccgac
      181 gttaatttaa gactggttaa gttttagagc tagaaatagc aagttaaaat aaggctagtc
      241 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt gataaaacga aaggcccagt
      301 ctttcgactg agcctttcgt tttataaacg ggtcttgagg ggttttttaa gcttctgcag
      361 ctggatccat ggatatctag atttccgcaa aataaaaaag gggacctcta gggtccccaa
      421 ttaattagct aaagccgcta gcccatggga attcctgcag cccgggggat ccactagttc
      481 tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacgcgc
      541 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      601 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      661 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa gcgttaatat
      721 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccgt
      781 actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa
      841 cgcctggttg ctacgcctga ataagtgata ataagcggat gaatggcaga aattcgaaag
      901 caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt atgtctattg
      961 ctggtttacc ggtttattga ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg
     1021 aggccagttt gctcaggctc tccccgtgga ggtaataatt gacgatatga tcatttattc
     1081 tgcctcccag agcctgataa aaacggtgaa tccgttagcg aggtgccgcc ggcttccatt
     1141 caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat
     1201 agggcggcga ggcggctaca gccgatagtc tggaacagcg cacttacggg ttgctgcgca
     1261 acccaagtgc taccggcgcg gcagcgtgac ccgtgtcggc ggctccaacg gctcgccatc
     1321 gtccagaaaa cacggctcat cgggcatcgg caggcgctgc tgcccgcgcc gttcccattc
     1381 ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc ggaatgccgg gctggctggg
     1441 cggctcctcg ccggggccgg tcggtagttg ctgctcgccc ggatacaggg tcgggatgcg
     1501 gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg tcgtgatcaa ccaccacggc
     1561 ggcactgaac accgacaggc gcaactggtc gcggggctgg ccccacgcca cgcggtcatt
     1621 gaccacgtag gccgacacgg tgccggggcc gttgagcttc acgacggaga tccagcgctc
     1681 ggccaccaag tccttgactg cgtattggac cgtccgcaaa gaacgtccga tgagcttgga
     1741 aagtgttttc tggctgacca ccacggcgtt ctggtggccc atctgcgcca cgaggtgatg
     1801 cagcagcatt gccgccgtgg gtttcctcgc aataagcccg gcccacgcct catgcgcttt
     1861 gcgttccgtt tgcacccagt gaccgggctt gttcttggct tgaatgccga tttctctgga
     1921 ctgcgtggcc atgcttatct ccatgcggta ggggtgccgc acggttgcgg caccatgcgc
     1981 aatcagctgc aacttttcgg cagcgcgaca acaattatgc gttgcgtaaa agtggcagtc
     2041 aattacagat tttctttaac ctacgcaatg agctattgcg gggggtgccg caatgagctg
     2101 ttgcgtaccc ccctttttta agttgttgat ttttaagtct ttcgcatttc gccctatatc
     2161 tagttctttg gtgcccaaag aagggcaccc ctgcggggtt cccccacgcc ttcggcgcgg
     2221 ctccccctcc ggcaaaaagt ggcccctccg gggcttgttg atcgactgcg cggccttcgg
     2281 ccttgcccaa ggtggcgctg cccccttgga acccccgcac tcgccgccgt gaggctcggg
     2341 gggcaggcgg gcgggcttcg cccttcgact gcccccactc gcataggctt gggtcgttcc
     2401 aggcgcgtca aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact
     2461 tggtgtccaa ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa
     2521 aattaaaaaa attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg
     2581 gtcgaaggct gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc
     2641 tgtccatcag cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg
     2701 ccgctcgcgg ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag
     2761 ggcgaagccc ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt
     2821 gcgaagcaaa gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt
     2881 gcgctgcccc cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg
     2941 cgatcatgcg atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg
     3001 cctaccgcga gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact
     3061 gggcggccag cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc
     3121 ggcgcaagga cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga
     3181 agtcggccag ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg
     3241 acaagtacgg ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc
     3301 acatgaccgc gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca
     3361 tcggcaacaa agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc
     3421 tagggctgca acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct
     3481 acgaggccct ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc
     3541 cacgcgccta tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga
     3601 cgccggaagc cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc
     3661 tacaggccgc cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg
     3721 cccgagacct tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg
     3781 atatgcaggc caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac
     3841 agcgggaagt ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt
     3901 tccccgaaaa gtgccacctg ggatgaatgt cagctactgg gctatctgga caagggaaaa
     3961 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg
     4021 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     4081 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag
     4141 gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4201 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     4261 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     4321 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     4381 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     4441 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     4501 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     4561 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     4621 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     4681 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     4741 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     4801 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     4861 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     4921 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     4981 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5041 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5101 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc ccatgggcaa
     5161 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     5221 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtttt tatataagct
     5281 ttcagtcggc gcgaatgctc cacgccgcgc ccagcggcgc ggatccaaat aaaacgaaag
     5341 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcgctgca gccagccgtg
     5401 gtcgggcagc aggtagaagg cgccctcgtc ctgatcctcg cccgaaacct ccagcccccg
     5461 gtcgatggct tcgacgacat agccggccgc gcggcaggtg tcggtgagcg cggccagcag
     5521 ggcggcttcc tggtcagggg tcaggtcggt catgggc
//
