LOCUS       40924_3851        6935 bp DNA     circular SYN 13-MAY-2021
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 6935)
  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 6935)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6935)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "iScience.";
            date: "2018-08-31"; pages: "
            10.1016/j.isci.2018.07.024. Epub 2018 Aug 1"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6935
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     475..491
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        498..516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(607..2025)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        2487..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(2730..2746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2754..2770)
                     /label=lac operator
                     /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(2778..2808)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2823..2844)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3001..3105
                     /label=AmpR promoter
     CDS             3106..3963
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      complement(4304..4655)
                     /direction=LEFT
                     /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)"
     CDS             4669..5490
                     /codon_start=1
                     /label=pRO1600 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pRO1600 from Pseudomonas aeruginosa"
                     /translation="VASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPL"
     rep_origin      5959..6547
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     rep_origin      complement(join(6814..6935,1..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 aagggcctcg tgatacgcct atttttatag
     2941 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3001 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3061 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3121 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3181 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3241 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3301 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     3361 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3421 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3481 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3541 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3601 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     3661 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3721 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3781 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gatctcgcgg tatcattgca
     3841 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3901 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3961 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4021 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4081 cgtgagtttt cgttccactg agcgtcagac cccaattaca cgccactggc tgtgcttgct
     4141 ggggtgacgg tggcaacggt ggcggccttg ctgggctatc gcgttggaaa gaaacgaggg
     4201 aaaggggact gataaaccgg tcttagcccc tccccttggt gtccaaccgc tctgtaggcc
     4261 tctcaggcgc cgctggtgcc gctggttgga cgccaagggt gaatccgcct cgataccctg
     4321 attactcgct tcctgcgccc tctcaggcgg cgatagggga ctggtaaaac ggggattgcc
     4381 cagacgcctc ccccgcccct tcaggggcac aaatgcggcc ccaacggggc cacgtagtgg
     4441 tgcgtttttt gcgtttccac ccttttcttc cttttccctt ttaaaccttt taggacgtct
     4501 acaggccacg taatccgtgg cctgtagagt ttaaaaaggg acggatttgt tgccattaag
     4561 ggacggattt gttgttaaga agggacggat ttgttgttgt aaagggacgg atttgttgta
     4621 ttgtgggacg cagatacagt gtccccttat acacaaggaa tgtcgaacgt ggcctcaccc
     4681 ccaatggttt acaaaagcaa tgccctggtc gaggccgcgt atcgcctcag tgttcaggaa
     4741 cagcggatcg ttctggcctg tattagccag gtgaagagga gcgagcctgt caccgatgaa
     4801 gtgatgtatt cagtgacggc ggaggacata gcgacgatgg cgggtgtccc tatcgaatct
     4861 tcctacaacc agctcaaaga agcggccctg cgcctgaaac ggcgggaagt ccggttaacc
     4921 caagagccca atggcaaggg gaaaagaccg agtgtgatga ttaccggctg ggtgcaaaca
     4981 atcatctacc gggagggtga gggccgtgta gaactcaggt tcaccaaaga catgctgccg
     5041 tacctgacgg aactcaccaa acagttcacc aaatacgcct tggctgacgt ggccaagatg
     5101 gacagcaccc acgcgatcag gctttacgag ctgctcatgc aatgggacag catcggccag
     5161 cgcgaaatag aaattgacca gctgcgaaag tggtttcaac tggaaggccg gtatccctcg
     5221 atcaaggact tcaagttgcg agtgcttgat ccagccgtga cgcagatcaa cgagcacagc
     5281 ccgctacagg tggagtgggc gcagcgaaag accgggcgca aggtcacaca tctgttgttc
     5341 agttttggac cgaagaagcc cgccaaggcg gtgggtaagg ccccagcgaa gcgcaaggcc
     5401 gggaagattt cagatgctga gatcgcgaaa caggctcgcc ctggtgagac atgggaagcg
     5461 gcccgcgctc gactaaccca gatgccgctt gcgcaaacta ttaactggcg aactacttac
     5521 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     5581 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     5641 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     5701 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     5761 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     5821 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     5881 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5941 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6001 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6061 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     6121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     6481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     6541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     6781 cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat attttgttaa
     6841 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     6901 aaatccctta taaatcaaaa gaatagaccg agata
//
