LOCUS       sequence-269540-        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 
            cpaA and second one targets blaA (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)_cpaA-
            Pcons...
FEATURES             Location/Qualifiers
     source          1..6016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      193..236
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      393..439
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      865..908
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(928..947)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(929..947)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(957..974)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(957..973)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(966..988)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(1682..2344)
                     /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      2345..3116
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4596..5390
                     /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
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCATVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4650..4669)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5260..5279
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      5738..5784
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cgcgcccgtg aggccgagga tttcgcgctg gtcagacaac ctacttgccg tccccacatg
       61 ttagcgctac caagtgccga gagcgtgacg cgagtggctt gttttagagc tgtgaaaaca
      121 gcgagttaaa ataaggctta gtccgtactc aacttgaaaa ggtggcaccg attcggtgtt
      181 tttttttttt tgataaaacg aaaggcccag tctttcgact gagcctttcg ttttataaac
      241 gggtcttgag gggtttttta agcttctgca gctggatcca tggatatcta gatttccgca
      301 aaataaaaaa ggggacctct agggtcccca attaattagc taaagccgct agcccatggg
      361 aattcctgca gcccggggga tccactagtt ctcaaataaa acgaaaggct cagtcgaaag
      421 actgggcctt tcgttttatc tgttgtttgt cgctgcagcc agccgtggtc gggcagcagg
      481 tagaaggcgc cctcgtcctg atcctcgccc gaaacctcca gcccccggtc gatggcttcg
      541 acgacatagc cggccgcgcg gcaggtgtcg gtgagcgcgg ccagcagggc ggcttcctgg
      601 tcaggggtca ggtcggtcat gggcaagagg tccaggtcgt ggtttgtcgg cggcttctag
      661 catggaccgc ccgcgcccgt gaggccgagg atttcgcgct ggtcagacaa cctacttgcc
      721 gtccccacat gttagcgcta ccaagtgccg accaggatca ggcgcttcat ggttttagag
      781 ctgtgaaaac agcgagttaa aataaggctt agtccgtact caacttgaaa aggtggcacc
      841 gattcggtgt tttttttttt tttgataaaa cgaaaggccc agtctttcga ctgagccttt
      901 cgttttataa acgggtcttc caattcgccc tatagtgagt cgtattacgc gcgctcactg
      961 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     1021 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     1081 tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa
     1141 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gtactgcgat
     1201 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
     1261 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg
     1321 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta
     1381 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt
     1441 ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc
     1501 agagcctgat aaaaacggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     1561 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     1621 gaggcggcta cagccgatag tctggaacag cgcacttacg ggttgctgcg caacccaagt
     1681 gctaccggcg cggcagcgtg acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa
     1741 aacacggctc atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt
     1801 cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc gggctggctg ggcggctcct
     1861 cgccggggcc ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt
     1921 cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga
     1981 acaccgacag gcgcaactgg tcgcggggct ggccccacgc cacgcggtca ttgaccacgt
     2041 aggccgacac ggtgccgggg ccgttgagct tcacgacgga gatccagcgc tcggccacca
     2101 agtccttgac tgcgtattgg accgtccgca aagaacgtcc gatgagcttg gaaagtgtct
     2161 tctggctgac caccacggcg ttctggtggc ccatctgcgc cacgaggtga tgcagcagca
     2221 ttgccgccgt gggtttcctc gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg
     2281 tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg
     2341 ccatgcttat ctccatgcgg taggggtgcc gcacggttgc ggcaccatgc gcaatcagct
     2401 gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag tcaattacag
     2461 attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc tgttgcgtac
     2521 cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata tctagttctt
     2581 tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc ggctccccct
     2641 ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc ggccttgccc
     2701 aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg gggggcaggc
     2761 gggcgggctt cgcccttcga ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt
     2821 caaggccaag ccgctgcgcg gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc
     2881 aaccggcaag cgaagcgcgc aggccgcagg ccggatgctt ttccccagag aaaattaaaa
     2941 aaattgatgg ggcaaggccg caggccgcgc agttggagcc ggtgggtatg tggtcgaagg
     3001 ctgggtagcc ggtgggcaat ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc
     3061 agcttgtcca gcagggttgt ccacgtgccg agcgaagcga gccagccggt ggccgctcgc
     3121 ggccatcgtc cacatatcca cgggctggca agggagcgca gcgaccgcgc agggcgaagc
     3181 ccggagagca agcccgtagg gcgccgcagc cgccgtaggc ggtcatgact ttgcgaagca
     3241 aagtctagtg agtatactca agcattgagt ggcccgccgg aggcaccgcc ttgcgctgcc
     3301 cccgtcgagc cggttggaca ccaaaaggga ggggcaggca tggcggcata cgcgatcatg
     3361 cgatgcaaga agctggcgaa aatgtgcaac gtggcggcca gtctcaagca cgcctaccgc
     3421 gagcgcgaga cgcccaacgc tgacgccagc aggacgccag agaacgagca ctgggcggcc
     3481 agcagcaccg atgaagcgat gggccgactg cgcgagttgc tgccagagaa gcggcgcaag
     3541 gacgctgtgt tggcggtcga gtacgtcatg acggccagcc cggaatggtg gaagtcggcc
     3601 agccaagaac agcaggcggc gttcttcgag aaggcgcaca agtggctggc ggacaagtac
     3661 ggggcggatc gcatcgtgac ggccagcatc caccgtgacg aaacaagccc gcacatgacc
     3721 gcgttcgtgg tgccgctgac gcaggacggc aggctgtcgg ccaaggagtt catcggcaac
     3781 aaagcgcaga tgacccgcga ccagaccacg tttgcggccg ctgtggccga tctagggctg
     3841 caacggggca tcgagggcag caaggcacgt cacacgcgca ttcaggcgtt ctacgaggcc
     3901 ctggagcggc caccagtggg ccacgtcacc atcagcccgc aagcggtcga gccacgcgcc
     3961 tatgcaccgc agggattggc cgaaaagctg ggaatctcaa agcgcgttga gacgccggaa
     4021 gccgtggccg accggctgac aaaagcggtt cggcaggggt atgagcctgc cctacaggcc
     4081 gccacaggag cgcgtgagat gcgcaagaag gccgatcaag cccaagagac ggcccgagac
     4141 cttcgggagc gcctgaagcc cgttctggac gccctggggc cgttgaatcg ggatatgcag
     4201 gccaaggccg ccgcgatcat caaggccgtg ggcgaaaagc tgctgacgga acagcgggaa
     4261 gtccagcgcc agaaacaggc ccagcgccag caggaacgcg ggcgcgcaca tttccccgaa
     4321 aagtgccacc tgggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
     4381 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt
     4441 tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
     4501 cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa
     4561 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     4621 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     4681 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     4741 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt
     4801 ggctggccac gacgggcgtt ccttgcgcaa ctgtgctcga cgttgtcact gaagcgggaa
     4861 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     4921 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     4981 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     5041 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     5101 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg
     5161 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     5221 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     5281 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     5341 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     5401 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     5461 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     5521 cctccagcgc ggggatctca tgctggagtt cttcgcccac ccccatgggc aaatattata
     5581 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     5641 gcttccatgt cggcagaatg cttaatgaat tacaacagtt tttatataag ctttcagtcg
     5701 gcgcgaatgc tccacgccgc gcccagcggc gcggatccaa ataaaacgaa aggctcagtc
     5761 gaaagactgg gcctttcgtt ttatctgttg tttgtcgctg cagccagccg tggtcgggca
     5821 gcaggtagaa ggcgccctcg tcctgatcct cgcccgaaac ctccagcccc cggtcgatgg
     5881 cttcgacgac atagccggcc gcgcggcagg tgtcggtgag cgcggccagc agggcggctt
     5941 cctggtcagg ggtcaggtcg gtcatgggca agaggtccag gtcgtggttt gtcggcggct
     6001 tctagcatgg accgcc
//
