LOCUS       sequence-257370-        5581 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Derived from pSCB2-sgRNA. 20 nucleotide binding sequence of sgRNA 
            removed by inverse PCR. Used as a negative control..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5581)
  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 5581)
  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-guideless
FEATURES             Location/Qualifiers
     source          1..5581
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(67..72,73..89,90..95))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        161..194
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        195..270
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(317..334)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      487..573
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      665..692
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(1158..1820)
                     /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      1821..2592
                     /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     3850..3868
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3985..4087
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(4154..4173)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(5043..5062)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(5086..5322)
                     /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 ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgagcgcat acgctacttg
       61 cattacagtt tacgaaccga acaggcttat gtcaactggg ttcgtgcggg tacctattat
      121 ttcctgtcag taacgagaag gtcgcgaatt ctaaagatct ttgacagcta gctcagtcct
      181 aggtataata ctaggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
      241 caacttgaaa aagtggcacc gagtcggtgc tttttttgaa gcttggctgt tttggcggat
      301 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
      361 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
      421 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
      481 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
      541 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
      601 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
      661 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tgttattggt
      721 cgacttaaac gcctggtgct acgcctgaat aagtgataat aagcggatga atggcagaaa
      781 ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat
      841 gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca
      901 aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc
      961 atttattctg cctcccagag cctgataaaa acggtgaatc cgttagcgag gtgccgccgg
     1021 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     1081 caaggtatag ggcggcgagg cggctacagc cgatagtctg gaacagcgca cttacgggtt
     1141 gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc gtgtcggcgg ctccaacggc
     1201 tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt
     1261 tcccattcct ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc
     1321 tggctgggcg gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg atacagggtc
     1381 gggatgcggc gcaggtcgcc atgccccaac agcgattcgt cctggtcgtc gtgatcaacc
     1441 accacggcgg cactgaacac cgacaggcgc aactggtcgc ggggctggcc ccacgccacg
     1501 cggtcattga ccacgtaggc cgacacggtg ccggggccgt tgagcttcac gacggagatc
     1561 cagcgctcgg ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg
     1621 agcttggaaa gtgtcttctg gctgaccacc acggcgttct ggtggcccat ctgcgccacg
     1681 aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa taagcccggc ccacgcctca
     1741 tgcgctttgc gttccgtttg cacccagtga ccgggcttgt tcttggcttg aatgccgatt
     1801 tctctggact gcgtggccat gcttatctcc atgcggtagg ggtgccgcac ggttgcggca
     1861 ccatgcgcaa tcagctgcaa cttttcggca gcgcgacaac aattatgcgt tgcgtaaaag
     1921 tggcagtcaa ttacagattt tctttaacct acgcaatgag ctattgcggg gggtgccgca
     1981 atgagctgtt gcgtaccccc cttttttaag ttgttgattt ttaagtcttt cgcatttcgc
     2041 cctatatcta gttctttggt gcccaaagaa gggcacccct gcggggttcc cccacgcctt
     2101 cggcgcggct ccccctccgg caaaaagtgg cccctccggg gcttgttgat cgactgcgcg
     2161 gccttcggcc ttgcccaagg tggcgctgcc cccttggaac ccccgcactc gccgccgtga
     2221 ggctcggggg gcaggcgggc gggcttcgcc cttcgactgc ccccactcgc ataggcttgg
     2281 gtcgttccag gcgcgtcaag gccaagccgc tgcgcggtcg ctgcgcgagc cttgacccgc
     2341 cttccacttg gtgtccaacc ggcaagcgaa gcgcgcaggc cgcaggccgg aggcttttcc
     2401 ccagagaaaa ttaaaaaaat tgatggggca aggccgcagg ccgcgcagtt ggagccggtg
     2461 ggtatgtggt cgaaggctgg gtagccggtg ggcaatccct gtggtcaagc tcgtgggcag
     2521 gcgcagcctg tccatcagct tgtccagcag ggttgtccac gggccgagcg aagcgagcca
     2581 gccggtggcc gctcgcggcc atcgtccaca tatccacggg ctggcaaggg agcgcagcga
     2641 ccgcgcaggg cgaagcccgg agagcaagcc cgtagggcgc cgcagccgcc gtaggcggtc
     2701 acgactttgc gaagcaaagt ctagtgagta tactcaagca ttgagtggcc cgccggaggc
     2761 accgccttgc gctgcccccg tcgagccggt tggacaccaa aagggagggg caggcatggc
     2821 ggcatacgcg atcatgcgat gcaagaagct ggcgaaaatg ggcaacgtgg cggccagtct
     2881 caagcacgcc taccgcgagc gcgagacgcc caacgctgac gccagcagga cgccagagaa
     2941 cgagcactgg gcggccagca gcaccgatga agcgatgggc cgactgcgcg agttgctgcc
     3001 agagaagcgg cgcaaggacg ctgtgttggc ggtcgagtac gtcatgacgg ccagcccgga
     3061 atggtggaag tcggccagcc aagaacagca ggcggcgttc ttcgagaagg cgcacaagtg
     3121 gctggcggac aagtacgggg cggatcgcat cgtgacggcc agcatccacc gtgacgaaac
     3181 cagcccgcac atgaccgcgt tcgtggtgcc gctgacgcag gacggcaggc tgtcggccaa
     3241 ggagttcatc ggcaacaaag cgcagatgac ccgcgaccag accacgtttg cggccgctgt
     3301 ggccgatcta gggctgcaac ggggcatcga gggcagcaag gcacgtcaca cgcgcattca
     3361 ggcgttctac gaggccctgg agcggccacc agtgggccac gtcaccatca gcccgcaagc
     3421 ggtcgagcca cgcgcctatg caccgcaggg attggccgaa aagctgggaa tctcaaagcg
     3481 cgttgagacg ccggaagccg tggccgaccg gctgacaaaa gcggttcggc aggggtatga
     3541 gcctgcccta caggccgccg caggagcgcg tgagatgcgc aagaaggccg atcaagccca
     3601 agagacggcc cgagaccttc gggagcgcct gaagcccgtt ctggacgccc tggggccgtt
     3661 gaatcgggat atgcaggcca aggccgccgc gatcatcaag gccgtgggcg aaaagctgct
     3721 gacggaacag cgggaagtcc agcgccagaa acaggcccag cgccagcagg aacgcgggcg
     3781 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     3841 acctataaaa ataggcgtat cacgaggccc tttcgtcttc gaataaatac ctgtgacgga
     3901 agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc
     3961 caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa
     4021 tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga
     4081 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     4141 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     4201 gctggatatt acggcctttt tagcttttgc cattctcacc ggattcagtc gtcactcatg
     4261 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     4321 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     4381 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     4441 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta
     4501 attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat
     4561 aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg
     4621 ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat
     4681 caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc
     4741 agcaacacct tcttcacgag gcagacctca gcgccagaag gccgccagag aggccgagcg
     4801 cggccgtgag gcttggacgc tagggcaggg catgaaaaag cccgtagcgg gctgctacgg
     4861 gcgtctgacg cggtggaaag ggggagggga tgttgtttaa atggctctgc tgtagtgagt
     4921 ggtctacggg gtctgacgct cagtggaacg aaatcgatga taagctgtca aacatgagca
     4981 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     5041 cgcctatatc gccgacatca ccgatgggag atcctaagat atcgcttagg ccacacgttc
     5101 aagtgcagcc acaggataaa tttgcactga gcctgggtgg gattcggact cgaccgcata
     5161 gccttcagga gtgagttttg tgcaatacca accgacgact tgaccctgcc aagcggcacc
     5221 agatttcttg cgtacgcgat cccctaagcc aaaggtggca ctcaggggaa gcgcaaactg
     5281 ccctgcaacg ggagcgttgg cttcatcgct actttgaccc atgtcgaatc cttcttgtga
     5341 atctattatg gcgacaagca aatcgagctc tgactgccta ccccacaaca actatcagaa
     5401 agcaccagca caacggctgc ctaactttgt tttagggcga ctgccctgct gcgtaacatc
     5461 gttgctgctc cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc
     5521 ccgaggcata gactgtacaa aaaaacagtc ataacaagcc atgaaaaccg ccactgcgcc
     5581 g
//
