LOCUS       sequence-401033-        6711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A sgRNA expression plasmid for genome editing in Burkholderia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6711)
  AUTHORS   Ferreira MR, Queiroga V, Moreira LM
  TITLE     Genomic editing in Burkholderia multivorans by CRISPR/Cas9.
  JOURNAL   Appl Environ Microbiol. 2024 Feb 21;90(2):e0225023. doi: 
            10.1128/aem.02250-23. Epub 2024 Feb 2.
  PUBMED    38299816
REFERENCE   2  (bases 1 to 6711)
  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: "Appl 
            Environ Microbiol."; date: "2024-02-21"; volume: "90(2):e0225023. 
            doi"; pages: " 10.1128/aem.02250-23. Epub 2024 Feb 2"
COMMENT     Sequence Label: pACRISPR-KM
FEATURES             Location/Qualifiers
     source          1..6711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..37)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     228..247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     460..482
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     474..491
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     475..491
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     498..517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        498..516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     546..565
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(604..1938,1939..2025))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     regulatory      2462..2471
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        join(2487..2492,2493..2509,2510..2516)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     misc_RNA        2539..2614
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2694..2712)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2695..2712)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2730..2746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2730..2746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2743..2765)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2754..2770
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2778..2784,2785..2802,2803..2808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2823..2844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             3172..3966
                     /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(3226..3245)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3836..3855
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      4080..4431
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     rep_origin      5735..6323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6224..6243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6477..6494
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6590..6711,1..334))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
       61 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      121 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      181 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      241 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
      301 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
      361 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      421 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      481 cgacggccag tgaattgtaa tacgactcac tatagggcga attcgagctc ggtacccggg
      541 gatcccggca tcagagcaga ttgtactgag agtgcaccat aatcggcatt ttcttttgcg
      601 tttttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga caacagatgt
      661 tttcttgcct ttgatgttca gcaggaagct aggcgcaaac gttgattgtt tgtctgcgta
      721 gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg cttgaggtac
      781 agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt ttaacacaag
      841 gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat aaccaagcat
      901 gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg agtcagtgaa
      961 cagataccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat ctgttactgt
     1021 gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt ttagctcaat
     1081 cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt gcagaagttt
     1141 ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttgt taaataaaga
     1201 ttcttcgcct tggtagccat cttcagttcc agtgtttgct tcaaatacta agtatttgtg
     1261 gcctttatct tctacgtagt gaggatctct cagcgtatgg ttgtcgcctg agctgtagtt
     1321 gccttcatcg atgaactgct gtacattttg atacgttttt ccgtcaccgt caaagattga
     1381 tttataatcc tctacaccgt tgatgttcaa agagctgtct gatgctgata cgttaacttg
     1441 tgcagttgtc agtgtttgtt tgccgtaatg tttaccggag aaatcagtgt agaataaacg
     1501 gatttttccg tcagatgtaa atgtggctga acctgaccat tcttgtgttt ggtcttttag
     1561 gatagaatca tttgcatcga atttgtcgct gtctttaaag acgcggccag cgtttttcca
     1621 gctgtcaata gaagtttcgc cgactttttg atagaacatg taaatcgatg tgtcatccgc
     1681 atttttagga tctccggcta atgcaaagac gatgtggtag ccgtgatagt ttgcgacagt
     1741 gccgtcagcg ttttgtaatg gccagctgtc ccaaacgtcc aggccttttg cagaagagat
     1801 atttttaatt gtggacgaat cgaactcagg aacttgatat ttttcatttt tttgctgttc
     1861 agggatttgc agcatatcat ggcgtgtaat atgggaaatg ccgtatgttt ccttatatgg
     1921 cttttggttc gtttctttcg caaacgcttg agttgcgcct cctgccagca gtgcggtagt
     1981 aaaggttaat actgttgctt gttttgcaaa ctttttgatg ttcatcgttc atgtctcctt
     2041 ttttatgtac tgtgttagcg gtctgcttct tccagccctc ctgtttgaag atggcaagtt
     2101 agttacgcac aataaaaaaa gacctaaaat atgtaagggg tgacgccaaa gtatacactt
     2161 tgccctttac acattttagg tcttgcctgc tttatcagta acaaacccgc gcgatttact
     2221 tttcgacctc attctattag actctcgttt ggattgcaac tggtctattt tcctcttttg
     2281 tttgatagaa aatcataaaa ggatttgcag actacgggcc taaagaacta aaaaatctat
     2341 ctgtttcttt tcattctctg tattttttat agtttctgtt gcatgggcat aaagttgcgt
     2401 cgacgccttg ctcaaattgg aatcaggttt gtgccaatac cagtagaaac agacgaagaa
     2461 tccatggcgg ccgctgtcat gggcccttga caattaatca tccggctcgt ataatgtgtg
     2521 gcgagaccat tggtctcagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgagatctgt ccatacccat
     2641 ggtctagaat gctcgagact tttcatactc ccgccattca gaagcttgag tattctatag
     2701 tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2761 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2821 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2881 aacctgtcgt gccagaattc ttgaagacga aaggatgtca gctactgggc tatctggaca
     2941 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag
     3001 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct
     3061 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga
     3121 tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3181 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3241 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3301 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact ccaagacgag
     3361 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3421 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3481 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3541 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3601 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3661 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc ggatgcccga cggcgaggat
     3721 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3781 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     3841 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     3901 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     3961 ttctgagcgg gactctgggg ttcgaaatga caccggtctt agcccctccc cttggtgtcc
     4021 aaccgctctg taggcctctc aggcgccgct ggtgccgctg gttggacgcc aagggtgaat
     4081 ccgcctcgat accctgatta ctcgcttcct gcgccctctc aggcggcgat aggggactgg
     4141 taaaacgggg attgcccaga cgcctccccc gccccttcag gggcacaaat gcggccccaa
     4201 cggggccacg tagtggtgcg ttttttgcgt ttccaccctt ttcttccttt tcccttttaa
     4261 accttttagg acgtctacag gccacgtaat ccgtggcctg tagagtttaa aaagggacgg
     4321 atttgttgcc attaagggac ggatttgttg ttaagaaggg acggatttgt tgttgtaaag
     4381 ggacggattt gttgtattgt gggacgcaga tacagtgtcc ccttatacac aaggaatgtc
     4441 gaacgtggcc tcacccccaa tggtttacaa aagcaatgcc ctggtcgagg ccgcgtatcg
     4501 cctcagtgtt caggaacagc ggatcgttct ggcctgtatt agccaggtga agaggagcga
     4561 gcctgtcacc gatgaagtga tgtattcagt gacggcggag gacatagcga cgatggcggg
     4621 tgtccctatc gaatcttcct acaaccagct caaagaagcg gccctgcgcc tgaaacggcg
     4681 ggaagtccgg ttaacccaag agcccaatgg caaggggaaa agaccgagtg tgatgattac
     4741 cggctgggtg caaacaatca tctaccggga gggtgagggc cgtgtagaac tcaggttcac
     4801 caaagacatg ctgccgtacc tgacggaact caccaaacag ttcaccaaat acgccttggc
     4861 tgacgtggcc aagatggaca gcacccacgc gatcaggctt tacgagctgc tcatgcaatg
     4921 ggacagcatc ggccagcgcg aaatagaaat tgaccagctg cgaaagtggt ttcaactgga
     4981 aggccggtat ccctcgatca aggacttcaa gttgcgagtg cttgatccag ccgtgacgca
     5041 gatcaacgag cacagcccgc tacaggtgga gtgggcgcag cgaaagaccg ggcgcaaggt
     5101 cacacatctg ttgttcagtt ttggaccgaa gaagcccgcc aaggcggtgg gtaaggcccc
     5161 agcgaagcgc aaggccggga agatttcaga tgctgagatc gcgaaacagg ctcgccctgg
     5221 tgagacatgg gaagcggccc gcgctcgact aacccagatg ccgcttgcgc aaactattaa
     5281 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5341 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5401 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5461 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5521 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5581 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5641 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5701 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5761 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5821 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5881 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5941 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6001 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6061 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6121 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6181 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6241 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6301 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6361 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6421 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6481 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     6541 gcggtatttc acaccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     6601 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     6661 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat a
//
