LOCUS       sequence-256110-        5601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derived from pSCB2-sgRNA. 20 nucleotide sequence added as sgRNA 
            binding region by inverse PCR. Used as a negative control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5601)
  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 5601)
  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: pgRNA-non-target
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(245..250,251..267,268..273))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        339..373
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        393..468
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(515..532)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      685..771
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      863..890
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1356..2018)
                     /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      2019..2790
                     /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     4048..4066
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4183..4285
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4352..4371)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(5241..5260)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5284..5520)
                     /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"
ORIGIN
        1 accagcacaa cggctgccta actttgtttt agggcgactg ccctgctgcg taacatcgtt
       61 gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg
      121 aggcatagac tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt
      181 accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca
      241 ttacagttta cgaaccgaac aggcttatgt caactgggtt cgtgcgggta cctattattt
      301 cctgtcagta acgagaaggt cgcgaattct aaagatcttt gacagctagc tcagtcctag
      361 gtataatact agtcgacgaa tgccaagcga tagttttaga gctagaaata gcaagttaaa
      421 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt tttttgaagc
      481 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
      541 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
      601 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
      661 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
      721 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
      781 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
      841 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
      901 caaactcttg ttattggtcg acttaaacgc ctggtgctac gcctgaataa gtgataataa
      961 gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt
     1021 cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac cggaagcagt
     1081 gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc cgtggaggta
     1141 ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac ggtgaatccg
     1201 ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac
     1261 gcaacgcggg gaggcagaca aggtataggg cggcgaggcg gctacagccg atagtctgga
     1321 acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt
     1381 gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg catcggcagg
     1441 cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag gtctggttcc
     1501 atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc
     1561 tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag cgattcgtcc
     1621 tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg
     1681 ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc ggggccgttg
     1741 agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta ttggaccgtc
     1801 cgcaaagaac gtccgatgag cttggaaagt gtcttctggc tgaccaccac ggcgttctgg
     1861 tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt cctcgcaata
     1921 agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc gggcttgttc
     1981 ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat gcggtagggg
     2041 tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc gcgacaacaa
     2101 ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac gcaatgagct
     2161 attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt gttgattttt
     2221 aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg gcacccctgc
     2281 ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc cctccggggc
     2341 ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc
     2401 ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgccct tcgactgccc
     2461 ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct
     2521 gcgcgagcct tgacccgcct tccacttggt gtccaaccgg caagcgaagc gcgcaggccg
     2581 caggccggag gcttttcccc agagaaaatt aaaaaaattg atggggcaag gccgcaggcc
     2641 gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg caatccctgt
     2701 ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg tccagcaggg ttgtccacgg
     2761 gccgagcgaa gcgagccagc cggtggccgc tcgcggccat cgtccacata tccacgggct
     2821 ggcaagggag cgcagcgacc gcgcagggcg aagcccggag agcaagcccg tagggcgccg
     2881 cagccgccgt aggcggtcac gactttgcga agcaaagtct agtgagtata ctcaagcatt
     2941 gagtggcccg ccggaggcac cgccttgcgc tgcccccgtc gagccggttg gacaccaaaa
     3001 gggaggggca ggcatggcgg catacgcgat catgcgatgc aagaagctgg cgaaaatggg
     3061 caacgtggcg gccagtctca agcacgccta ccgcgagcgc gagacgccca acgctgacgc
     3121 cagcaggacg ccagagaacg agcactgggc ggccagcagc accgatgaag cgatgggccg
     3181 actgcgcgag ttgctgccag agaagcggcg caaggacgct gtgttggcgg tcgagtacgt
     3241 catgacggcc agcccggaat ggtggaagtc ggccagccaa gaacagcagg cggcgttctt
     3301 cgagaaggcg cacaagtggc tggcggacaa gtacggggcg gatcgcatcg tgacggccag
     3361 catccaccgt gacgaaacca gcccgcacat gaccgcgttc gtggtgccgc tgacgcagga
     3421 cggcaggctg tcggccaagg agttcatcgg caacaaagcg cagatgaccc gcgaccagac
     3481 cacgtttgcg gccgctgtgg ccgatctagg gctgcaacgg ggcatcgagg gcagcaaggc
     3541 acgtcacacg cgcattcagg cgttctacga ggccctggag cggccaccag tgggccacgt
     3601 caccatcagc ccgcaagcgg tcgagccacg cgcctatgca ccgcagggat tggccgaaaa
     3661 gctgggaatc tcaaagcgcg ttgagacgcc ggaagccgtg gccgaccggc tgacaaaagc
     3721 ggttcggcag gggtatgagc ctgccctaca ggccgccgca ggagcgcgtg agatgcgcaa
     3781 gaaggccgat caagcccaag agacggcccg agaccttcgg gagcgcctga agcccgttct
     3841 ggacgccctg gggccgttga atcgggatat gcaggccaag gccgccgcga tcatcaaggc
     3901 cgtgggcgaa aagctgctga cggaacagcg ggaagtccag cgccagaaac aggcccagcg
     3961 ccagcaggaa cgcgggcgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     4021 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcga
     4081 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga
     4141 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg
     4201 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttatcgag
     4261 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     4321 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     4381 ctataaccag accgttcagc tggatattac ggccttttta gcttttgcca ttctcaccgg
     4441 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     4501 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     4561 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     4621 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     4681 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     4741 acggcggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga tcacgcatct
     4801 tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca actggtccac
     4861 ctacaacaaa gctctcatca accgtggctc cctcactttc tggctggatg atggggcgat
     4921 tcaggcctgg tatgagtcag caacaccttc ttcacgaggc agacctcagc gccagaaggc
     4981 cgccagagag gccgagcgcg gccgtgaggc ttggacgcta gggcagggca tgaaaaagcc
     5041 cgtagcgggc tgctacgggc gtctgacgcg gtggaaaggg ggaggggatg ttgtttaaat
     5101 ggctctgctg tagtgagtgg tctacggggt ctgacgctca gtggaacgaa atcgatgata
     5161 agctgtcaaa catgagcaga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     5221 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatgggagat cctaagatat
     5281 cgcttaggcc acacgttcaa gtgcagccac aggataaatt tgcactgagc ctgggtggga
     5341 ttcggactcg accgcatagc cttcaggagt gagttttgtg caataccaac cgacgacttg
     5401 accctgccaa gcggcaccag atttcttgcg tacgcgatcc cctaagccaa aggtggcact
     5461 caggggaagc gcaaactgcc ctgcaacggg agcgttggct tcatcgctac tttgacccat
     5521 gtcgaatcct tcttgtgaat ctattatggc gacaagcaaa tcgagctctg actgcctacc
     5581 ccacaacaac tatcagaaag c
//
