LOCUS       sequence-267409-        6016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of two sgRNA from Streptococcus thermophilus #3 each 
            express from its own constitutive promoter; here first one targets 
            blaA and second one targets cpaA (from Caulobacter crescentus).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6016)
  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 6016)
  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(Sth3)_blaA-
            Pcons...
FEATURES             Location/Qualifiers
     source          1..6016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      356..399
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(419..438)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(420..438)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(448..465)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(448..464)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(457..479)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1173..1835)
                     /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      1836..2607
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4087..4881
                     /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(4141..4160)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4751..4770
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5229..5275
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5700..5743
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5900..5946
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgaaacctcc agcccccggt cgatggcttc gacgacatag ccggccgcgc ggcaggtgtc
       61 ggtgagcgcg gccagcaggg cggcttcctg gtcaggggtc aggtcggtca tgggcaagag
      121 gtccaggtcg tggtttgtcg gcggcttcta gcatggaccg cccgcgcccg tgaggccgag
      181 gatttcgcgc tggtcagaca acctacttgc cgtccccaca tgttagcgct accaagtgcc
      241 gagagcgtga cgcgagtggc ttgttttaga gctgtgaaaa cagcgagtta aaataaggct
      301 tagtccgtac tcaacttgaa aaggtggcac cgattcggtg tttttttttt ttttgataaa
      361 acgaaaggcc cagtctttcg actgagcctt tcgttttata aacgggtctt ccaattcgcc
      421 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
      481 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
      541 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
      601 atggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      661 tcatttttta accaataggc cgtactgcga tgagtggcag ggcggggcgt aattttttta
      721 aggcagttat tggtgccctt aaacgcctgg ttgctacgcc tgaataagtg ataataagcg
      781 gatgaatggc agaaattcga aagcaaattc gacccggtcg tcggttcagg gcagggtcgt
      841 taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg aagcagtgtg
      901 accgtgtgct tctcaaatgc ctgaggccag tttgctcagg ctctccccgt ggaggtaata
      961 attgacgata tgatcattta ttctgcctcc cagagcctga taaaaacggt gaatccgtta
     1021 gcgaggtgcc gccggcttcc attcaggtcg aggtggcccg gctccatgca ccgcgacgca
     1081 acgcggggag gcagacaagg tatagggcgg cgaggcggct acagccgata gtctggaaca
     1141 gcgcacttac gggttgctgc gcaacccaag tgctaccggc gcggcagcgt gacccgtgtc
     1201 ggcggctcca acggctcgcc atcgtccaga aaacacggct catcgggcat cggcaggcgc
     1261 tgctgcccgc gccgttccca ttcctccgtt tcggtcaagg ctggcaggtc tggttccatg
     1321 cccggaatgc cgggctggct gggcggctcc tcgccggggc cggtcggtag ttgctgctcg
     1381 cccggataca gggtcgggat gcggcgcagg tcgccatgcc ccaacagcga ttcgtcctgg
     1441 tcgtcgtgat caaccaccac ggcggcactg aacaccgaca ggcgcaactg gtcgcggggc
     1501 tggccccacg ccacgcggtc attgaccacg taggccgaca cggtgccggg gccgttgagc
     1561 ttcacgacgg agatccagcg ctcggccacc aagtccttga ctgcgtattg gaccgtccgc
     1621 aaagaacgtc cgatgagctt ggaaagtgtc ttctggctga ccaccacggc gttctggtgg
     1681 cccatctgcg ccacgaggtg atgcagcagc attgccgccg tgggtttcct cgcaataagc
     1741 ccggcccacg cctcatgcgc tttgcgttcc gtttgcaccc agtgaccggg cttgttcttg
     1801 gcttgaatgc cgatttctct ggactgcgtg gccatgctta tctccatgcg gtaggggtgc
     1861 cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta
     1921 tgcgttgcgt aaaagtggca gtcaattaca tattttcttt aacctacgca atgagctatt
     1981 gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag
     2041 tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg
     2101 gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg
     2161 ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg
     2221 cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgcccttcg actgccccca
     2281 ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg
     2341 cgagccttga cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg caggccgcag
     2401 gccggatgct tttccccaga gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg
     2461 cagttggagc cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt
     2521 caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc agcagggttg tccacgtgcc
     2581 gagcgaagcg agccagccgg tggccgctcg cggccatcgt ccacatatcc acgggctggc
     2641 aagggagcgc agcgaccgcg cagggcgaag cccggagagc aagcccgtag ggcgccgcag
     2701 ccgccgtagg cggtcatgac tttgcgaagc aaagtctagt gagtatactc aagcattgag
     2761 tggcccgccg gaggcaccgc cttgcgctgc ccccgtcgag ccggttggac accaaaaggg
     2821 aggggcaggc atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgtgcaa
     2881 cgtggcggcc agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag
     2941 caggacgcca gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact
     3001 gcgcgagttg ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat
     3061 gacggccagc ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga
     3121 gaaggcgcac aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat
     3181 ccaccgtgac gaaacaagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg
     3241 caggctgtcg gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac
     3301 gtttgcggcc gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg
     3361 tcacacgcgc attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac
     3421 catcagcccg caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct
     3481 gggaatctca aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt
     3541 tcggcagggg tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa
     3601 ggccgatcaa gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga
     3661 cgccctgggg ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt
     3721 gggcgaaaag ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca
     3781 gcaggaacgc gggcgcgcac atttccccga aaagtgccac ctgggatgaa tgtcagctac
     3841 tgggctatct ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg
     3901 cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc ggaattgcca
     3961 gctggggcgc cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttcttg
     4021 ccgccaagga tctgatggcg caggggatca agatctgatc aagagacagg atgaggatcg
     4081 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4141 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4201 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4261 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4321 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4381 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4441 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4501 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4561 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4621 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4681 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4741 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4801 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4861 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4921 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4981 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5041 tcttcgccca cccccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg
     5101 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa
     5161 ttacaacagt ttttatataa gctttcagtc ggcgcgaatg ctccacgccg cgcccagcgg
     5221 cgcggatcca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     5281 gtttgtcgct gcagccagcc gtggtcgggc agcaggtaga aggcgccctc gtcctgatcc
     5341 tcgcccgaaa cctccagccc ccggtcgatg gcttcgacga catagccggc cgcgcggcag
     5401 gtgtcggtga gcgcggccag cagggcggct tcctggtcag gggtcaggtc ggtcatgggc
     5461 aagaggtcca ggtcgtggtt tgtcggcggc ttctagcatg gaccgcccgc gcccgtgagg
     5521 ccgaggattt cgcgctggtc agacaaccta cttgccgtcc ccacatgtta gcgctaccaa
     5581 gtgccgacca ggatcaggcg cttcatggtt ttagagctgt gaaaacagcg agttaaaata
     5641 aggcttagtc cgtactcaac ttgaaaaggt ggcaccgatt cggtgttttt ttttttttga
     5701 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tataaacggg tcttgagggg
     5761 ttttttaagc ttctgcagct ggatccatgg atatctagat ttccgcaaaa taaaaaaggg
     5821 gacctctagg gtccccaatt aattagctaa agccgctagc ccatgggaat tcctgcagcc
     5881 cgggggatcc actagttctc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     5941 ttttatctgt tgtttgtcgc tgcagccagc cgtggtcggg cagcaggtag aaggcgccct
     6001 cgtcctgatc ctcgcc
//
