LOCUS       sequence-257368-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid for guide sequence insertion via inverse PCR. 
            Derived from pSCrhaB2-gRNA (see paper)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  AUTHORS   Hogan AM, Rahman ASMZ, Lightly TJ, Cardona ST
  TITLE     A broad-host-range CRISPRi toolkit for silencing gene expression in 
            Burkholderia.
  JOURNAL   ACS Synth Biol. 2019 Sep 6. doi: 10.1021/acssynbio.9b00232.
  PUBMED    31491085
REFERENCE   2  (bases 1 to 5602)
  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"; journalName: "ACS Synth 
            Biol. 2019 Sep 6. doi: 10.1021/acssynbio.9b00232."
COMMENT     Sequence Label: pSCB2-sgRNA
FEATURES             Location/Qualifiers
     source          1..5602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..22)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      175..261
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      353..380
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(846..1508)
                     /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      1509..2280
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     primer_bind     3538..3556
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3673..3775
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3842..3861)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(4731..4750)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4774..5010)
                     /codon_start=1
                     /gene="dhfrIIb from plasmid R388"
                     /product="E. coli plasmid-associated dihydrofolate 
                     reductase"
                     /label=TpR
                     /note="confers resistance to trimethoprim"
                     /translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
                     YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
     promoter        complement(join(5336..5341,5342..5358,5359..5364))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        5430..5464
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        5485..5560
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
       61 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
      121 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
      181 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
      241 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
      301 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
      361 atcctgacgg atggcctttt tgcgtttcta caaactcttg ttattggtcg acttaaacgc
      421 ctggtgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa
      481 ttcgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgctgg
      541 tttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc
      601 cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcat ttattctgcc
      661 tcccagagcc tgataaaaac ggtgaatccg ttagcgaggt gccgccggct tccattcagg
      721 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
      781 cggcgaggcg gctacagccg atagtctgga acagcgcact tacgggttgc tgcgcaaccc
      841 aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc
      901 agaaaacacg gctcatcggg catcggcagg cgctgctgcc cgcgccgttc ccattcctcc
      961 gtttcggtca aggctggcag gtctggttcc atgcccggaa tgccgggctg gctgggcggc
     1021 tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat acagggtcgg gatgcggcgc
     1081 aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca
     1141 ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc
     1201 acgtaggccg acacggtgcc ggggccgttg agcttcacga cggagatcca gcgctcggcc
     1261 accaagtcct tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt
     1321 gtcttctggc tgaccaccac ggcgttctgg tggcccatct gcgccacgag gtgatgcagc
     1381 agcattgccg ccgtgggttt cctcgcaata agcccggccc acgcctcatg cgctttgcgt
     1441 tccgtttgca cccagtgacc gggcttgttc ttggcttgaa tgccgatttc tctggactgc
     1501 gtggccatgc ttatctccat gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc
     1561 agctgcaact tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt
     1621 acagattttc tttaacctac gcaatgagct attgcggggg gtgccgcaat gagctgttgc
     1681 gtacccccct tttttaagtt gttgattttt aagtctttcg catttcgccc tatatctagt
     1741 tctttggtgc ccaaagaagg gcacccctgc ggggttcccc cacgccttcg gcgcggctcc
     1801 ccctccggca aaaagtggcc cctccggggc ttgttgatcg actgcgcggc cttcggcctt
     1861 gcccaaggtg gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc
     1921 aggcgggcgg gcttcgccct tcgactgccc ccactcgcat aggcttgggt cgttccaggc
     1981 gcgtcaaggc caagccgctg cgcggtcgct gcgcgagcct tgacccgcct tccacttggt
     2041 gtccaaccgg caagcgaagc gcgcaggccg caggccggag gcttttcccc agagaaaatt
     2101 aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg
     2161 aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc
     2221 catcagcttg tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc
     2281 tcgcggccat cgtccacata tccacgggct ggcaagggag cgcagcgacc gcgcagggcg
     2341 aagcccggag agcaagcccg tagggcgccg cagccgccgt aggcggtcac gactttgcga
     2401 agcaaagtct agtgagtata ctcaagcatt gagtggcccg ccggaggcac cgccttgcgc
     2461 tgcccccgtc gagccggttg gacaccaaaa gggaggggca ggcatggcgg catacgcgat
     2521 catgcgatgc aagaagctgg cgaaaatggg caacgtggcg gccagtctca agcacgccta
     2581 ccgcgagcgc gagacgccca acgctgacgc cagcaggacg ccagagaacg agcactgggc
     2641 ggccagcagc accgatgaag cgatgggccg actgcgcgag ttgctgccag agaagcggcg
     2701 caaggacgct gtgttggcgg tcgagtacgt catgacggcc agcccggaat ggtggaagtc
     2761 ggccagccaa gaacagcagg cggcgttctt cgagaaggcg cacaagtggc tggcggacaa
     2821 gtacggggcg gatcgcatcg tgacggccag catccaccgt gacgaaacca gcccgcacat
     2881 gaccgcgttc gtggtgccgc tgacgcagga cggcaggctg tcggccaagg agttcatcgg
     2941 caacaaagcg cagatgaccc gcgaccagac cacgtttgcg gccgctgtgg ccgatctagg
     3001 gctgcaacgg ggcatcgagg gcagcaaggc acgtcacacg cgcattcagg cgttctacga
     3061 ggccctggag cggccaccag tgggccacgt caccatcagc ccgcaagcgg tcgagccacg
     3121 cgcctatgca ccgcagggat tggccgaaaa gctgggaatc tcaaagcgcg ttgagacgcc
     3181 ggaagccgtg gccgaccggc tgacaaaagc ggttcggcag gggtatgagc ctgccctaca
     3241 ggccgccgca ggagcgcgtg agatgcgcaa gaaggccgat caagcccaag agacggcccg
     3301 agaccttcgg gagcgcctga agcccgttct ggacgccctg gggccgttga atcgggatat
     3361 gcaggccaag gccgccgcga tcatcaaggc cgtgggcgaa aagctgctga cggaacagcg
     3421 ggaagtccag cgccagaaac aggcccagcg ccagcaggaa cgcgggcgcg cacatttccc
     3481 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3541 aggcgtatca cgaggccctt tcgtcttcga ataaatacct gtgacggaag atcacttcgc
     3601 agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca acttttggcg
     3661 aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc
     3721 actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgga
     3781 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     3841 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     3901 ggccttttta gcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac
     3961 ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg
     4021 gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc
     4081 cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat
     4141 tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac
     4201 actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt
     4261 tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa
     4321 gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc
     4381 cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc
     4441 ttcacgaggc agacctcagc gccagaaggc cgccagagag gccgagcgcg gccgtgaggc
     4501 ttggacgcta gggcagggca tgaaaaagcc cgtagcgggc tgctacgggc gtctgacgcg
     4561 gtggaaaggg ggaggggatg ttgtttaaat ggctctgctg tagtgagtgg tctacggggt
     4621 ctgacgctca gtggaacgaa atcgatgata agctgtcaaa catgagcaga tcctctacgc
     4681 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     4741 cgacatcacc gatgggagat cctaagatat cgcttaggcc acacgttcaa gtgcagccac
     4801 aggataaatt tgcactgagc ctgggtggga ttcggactcg accgcatagc cttcaggagt
     4861 gagttttgtg caataccaac cgacgacttg accctgccaa gcggcaccag atttcttgcg
     4921 tacgcgatcc cctaagccaa aggtggcact caggggaagc gcaaactgcc ctgcaacggg
     4981 agcgttggct tcatcgctac tttgacccat gtcgaatcct tcttgtgaat ctattatggc
     5041 gacaagcaaa tcgagctctg actgcctacc ccacaacaac tatcagaaag caccagcaca
     5101 acggctgcct aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctcca
     5161 taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga
     5221 ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct
     5281 gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagttt
     5341 acgaaccgaa caggcttatg tcaactgggt tcgtgcgggt acctattatt tcctgtcagt
     5401 aacgagaagg tcgcgaattc taaagatctt tgacagctag ctcagtccta ggtataatac
     5461 tagtaacttt cagtttagcg gtctgtttta gagctagaaa tagcaagtta aaataaggct
     5521 agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa gcttggctgt
     5581 tttggcggat gagagaagat tt
//
