LOCUS       sequence-213880-        6936 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A sgRNA expression plasmid for genome editing in Pseudomonas 
            aeruginosa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6936)
  AUTHORS   Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L, Wang Y, Ji Q
  TITLE     CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and 
            Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.
  JOURNAL   iScience. 2018 Aug 31;6:222-231. doi: 10.1016/j.isci.2018.07.024. 
            Epub 2018 Aug 1.
  PUBMED    30240613
REFERENCE   2  (bases 1 to 6936)
  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: "iScience.";
            date: "2018-08-31"; volume: "6:222-231. doi"; pages: " 
            10.1016/j.isci.2018.07.024. Epub 2018 Aug 1"
COMMENT     Sequence Label: pACRISPR
FEATURES             Location/Qualifiers
     source          1..6936
                     /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"
     promoter        complement(join(2779..2785,2786..2803,2804..2809))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2824..2845
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2916..2934)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3002..3106
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3107..3175,3176..3967)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3325..3344)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4305..4656
                     /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      5960..6548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6449..6468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6702..6719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6815..6936,1..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 aaattgtgta
     2761 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     2821 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     2881 aaacctgtcg tgccagaatt cttgaagacg aaagggcctc gtgatacgcc tatttttata
     2941 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     3001 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3061 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     3121 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     3181 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     3241 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     3301 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg
     3361 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     3421 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     3481 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     3541 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     3601 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc
     3661 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3721 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3781 tggctggttt attgctgata aatctggagc cggtgagcgt ggatctcgcg gtatcattgc
     3841 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3901 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3961 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     4021 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4081 acgtgagttt tcgttccact gagcgtcaga ccccaattac acgccactgg ctgtgcttgc
     4141 tggggtgacg gtggcaacgg tggcggcctt gctgggctat cgcgttggaa agaaacgagg
     4201 gaaaggggac tgataaaccg gtcttagccc ctccccttgg tgtccaaccg ctctgtaggc
     4261 ctctcaggcg ccgctggtgc cgctggttgg acgccaaggg tgaatccgcc tcgataccct
     4321 gattactcgc ttcctgcgcc ctctcaggcg gcgatagggg actggtaaaa cggggattgc
     4381 ccagacgcct cccccgcccc ttcaggggca caaatgcggc cccaacgggg ccacgtagtg
     4441 gtgcgttttt tgcgtttcca cccttttctt ccttttccct tttaaacctt ttaggacgtc
     4501 tacaggccac gtaatccgtg gcctgtagag tttaaaaagg gacggatttg ttgccattaa
     4561 gggacggatt tgttgttaag aagggacgga tttgttgttg taaagggacg gatttgttgt
     4621 attgtgggac gcagatacag tgtcccctta tacacaagga atgtcgaacg tggcctcacc
     4681 cccaatggtt tacaaaagca atgccctggt cgaggccgcg tatcgcctca gtgttcagga
     4741 acagcggatc gttctggcct gtattagcca ggtgaagagg agcgagcctg tcaccgatga
     4801 agtgatgtat tcagtgacgg cggaggacat agcgacgatg gcgggtgtcc ctatcgaatc
     4861 ttcctacaac cagctcaaag aagcggccct gcgcctgaaa cggcgggaag tccggttaac
     4921 ccaagagccc aatggcaagg ggaaaagacc gagtgtgatg attaccggct gggtgcaaac
     4981 aatcatctac cgggagggtg agggccgtgt agaactcagg ttcaccaaag acatgctgcc
     5041 gtacctgacg gaactcacca aacagttcac caaatacgcc ttggctgacg tggccaagat
     5101 ggacagcacc cacgcgatca ggctttacga gctgctcatg caatgggaca gcatcggcca
     5161 gcgcgaaata gaaattgacc agctgcgaaa gtggtttcaa ctggaaggcc ggtatccctc
     5221 gatcaaggac ttcaagttgc gagtgcttga tccagccgtg acgcagatca acgagcacag
     5281 cccgctacag gtggagtggg cgcagcgaaa gaccgggcgc aaggtcacac atctgttgtt
     5341 cagttttgga ccgaagaagc ccgccaaggc ggtgggtaag gccccagcga agcgcaaggc
     5401 cgggaagatt tcagatgctg agatcgcgaa acaggctcgc cctggtgaga catgggaagc
     5461 ggcccgcgct cgactaaccc agatgccgct tgcgcaaact attaactggc gaactactta
     5521 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5581 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5641 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5701 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5761 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5821 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5881 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6001 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6061 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     6121 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6181 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6241 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6301 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6361 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6421 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6481 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6541 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6601 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6661 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6721 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     6781 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
     6841 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     6901 aaaatccctt ataaatcaaa agaatagacc gagata
//
