LOCUS       sequence-203520-        4159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  W2.5-2 plasmid. Contains PLacO-sgRNA1, 
            PRha-sgRNA6-(sgRNA4-C16T-C18T) and is part of CAMERA 2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4159)
  AUTHORS   Tang W, Liu DR
  TITLE     Rewritable multi-event analog recording in bacterial and mammalian 
            cells.
  JOURNAL   Science. 2018 Feb 15. pii: science.aap8992. doi: 
            10.1126/science.aap8992.
  PUBMED    29449507
REFERENCE   2  (bases 1 to 4159)
  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: "Science. 
            2018 Feb 15. pii: science.aap8992. doi: 10.1126/science.aap8992."
COMMENT     Sequence Label: pWT050d
FEATURES             Location/Qualifiers
     source          1..4159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(39..777)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     misc_RNA        complement(917..992)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1013..1131)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             1277..2113
                     /codon_start=1
                     /gene="E. coli rhaS"
                     /product="positive regulator of the rhaB promoter
                     "
                     /label=rhaS
                     /note="activated by L-rhamnose"
                     /translation="MTVLHSVDFFPSGNASVAIEPRLPQADFPEHHHDFHEIVIVEHGT
                     GIHVFNGQPYTITGGTVCFVRDHDRHLYEHTDNLCLTNVLYRSPDRFQFLAGLNQLLPQ
                     ELDGQYPSHWRVNHSVLQQVRQLVAQMEQQEGENDLPSTASREILFMQLLLLLRKSSLQ
                     ENLENSASRLNLLLAWLEDHFADEVNWDAVADQFSLSLRTLHRQLKQQTGLTPQRYLNR
                     LRLMKARHLLRHSEASVTDIAYRCGFSDSNHFSTLFRREFNWSPRDIRQGRDGFLQ"
     CDS             2187..3035
                     /codon_start=1
                     /gene="E. coli rhaR"
                     /product="transcriptional activator of rhaR and rhaS"
                     /label=rhaR
                     /note="needed for rhamnose-dependent activation of the rhaB
                     promoter"
                     /translation="MAHQLKLLKDDFFASDQQAVAVADRYPQDVFAEHTHDFCELVIVW
                     RGNGLHVLNDRPYRITRGDLFYIHADDKHSYASVNDLVLQNIIYCPERLKLNLDWQGAI
                     PGFNASAGQPHWRLGSMGMAQARQVIGQLEHESSQHVPFANEMAELLFGQLVMLLNRHR
                     YTSDSLPPTSSETLLDKLITRLAASLKSPFALDKFCDEASCSERVLRQQFRQQTGMTIN
                     QYLRQVRVCHAQYLLQHSRLLISDISTECGFEDSNYFSVVFTRETGMTPSQWRHLNSQK
                     D"
     terminator      3043..3070
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3130..3175)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_RNA        complement(3183..3258)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    3291..3307
                     /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)."
     protein_bind    3314..3330
                     /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)."
     CDS             complement(3386..4045)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVTQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc gctgcggaca catacaaagt
       61 tacccacaga ttccgtggat aagcagggga ctaacatgtg aggcaaaaca gcagggccgc
      121 gccggtggcg tttttccata ggctccgccc tcctgccaga gttcacataa acagacgctt
      181 ttccggtgca tctgtgggag ccgtgaggct caaccatgaa tctgacagta cgggcgaaac
      241 ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg ctccctcttg cgctctcctg
      301 ttccgaccct gccgtttacc ggatacctgt tccgcctttc tcccttacgg gaagtgtggc
      361 gctttctcat agctcacaca ctggtatctc ggctcggtgt aggtcgttcg ctccaagctg
      421 ggctgtaagc aagaactccc cgttcagccc gactgctgcg ccttatccgg taactgttca
      481 cttgagtcca acccggaaaa gcacggtaaa acgccactgg cagcagccat tggtaactgg
      541 gagttcgcag aggatttgtt tagctaaaca cgcggttgct cttgaagtgt gcgccaaagt
      601 ccggctacac tggaaggaca gatttggttg ctgtgctctg cgaaagccag ttaccacggt
      661 taagcagttc cccaactgac ttaaccttcg atcaaaccac ctccccaggt ggttttttcg
      721 tttacagggc aaaagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      781 tctactgaac cgctctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
      841 agcccaggag gaaaaaaaac cccgccctgt caggggcggg gttttttttt atgttgaaaa
      901 tctccttcta aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc
      961 ttattttaac ttgctatttc tagctctaaa actcaaaacc accctgacct actacgacca
     1021 gtctaaaaag cgcctgaatt cgcgaccttc tcgttactga caggaaaatg ggccattggc
     1081 aaccagggaa agatgaacgt gatgatgttc acaatttgct gaattgtggt gatgtgatgc
     1141 tcaccgcatt tcctgaaaat tcacgctgta tcttgaaaaa tcgacgtttt ttacgtggtt
     1201 ttccgtcgaa aatttaaggt aagaacctga cctcgtgatt actatttcgc cgtgttgacg
     1261 acatcaggag gccagtatga ccgtattaca tagtgtggat ttttttccgt ctggtaacgc
     1321 gtccgtggcg atagaacccc ggctcccgca ggcggatttt cctgaacatc atcatgattt
     1381 tcatgaaatt gtgattgtcg aacatggcac gggtattcat gtgtttaatg ggcagcccta
     1441 taccatcacc ggtggcacgg tctgtttcgt acgcgatcat gatcggcatc tgtatgaaca
     1501 taccgataat ctgtgtctga ccaatgtgct gtatcgctcg ccggatcgat ttcagtttct
     1561 cgccgggctg aatcagttgc tgccacaaga gctggatggg cagtatccgt ctcactggcg
     1621 cgttaaccac agcgtattgc agcaggtgcg acagctggtt gcacagatgg aacagcagga
     1681 aggggaaaat gatttaccct cgaccgccag tcgcgagatc ttgtttatgc aattactgct
     1741 cttgctgcgt aaaagcagtt tgcaggagaa cctggaaaac agcgcatcac gtctcaactt
     1801 gcttctggcc tggctggagg accattttgc cgatgaggtg aattgggatg ccgtggcgga
     1861 tcaattttct ctttcactgc gtacgctaca tcggcagctt aagcagcaaa cgggactgac
     1921 gcctcagcga tacctgaacc gcctgcgact gatgaaagcc cgacatctgc tacgccacag
     1981 cgaggccagc gttactgaca tcgcctatcg ctgtggattc agcgacagta accacttttc
     2041 gacgcttttt cgccgagagt ttaactggtc accgcgtgat attcgccagg gacgggatgg
     2101 ctttctgcaa taacgcgaat cttctcaacg tatttgtacg ccatattgcg aataatcaac
     2161 ttcgttctct ggccgaggta gccacggtgg cgcatcagtt aaaacttctc aaagatgatt
     2221 tttttgccag cgaccagcag gcagtcgctg tggctgaccg ttatccgcaa gatgtctttg
     2281 ctgaacatac acatgatttt tgtgagctgg tgattgtctg gcgcggtaat ggcctgcatg
     2341 tactcaacga tcgcccttat cgcattaccc gtggcgatct cttttacatt catgctgacg
     2401 ataaacactc ctacgcttcc gttaacgatc tggttttgca gaatattatt tattgcccgg
     2461 agcgtctgaa gctgaatctt gactggcagg gggcgattcc gggatttaac gccagcgcag
     2521 ggcaaccaca ctggcgctta ggtagcatgg ggatggcgca ggcgcggcag gttatcggtc
     2581 agcttgagca tgaaagtagt cagcatgtgc cgtttgctaa cgaaatggct gagttgctgt
     2641 tcgggcagtt ggtgatgttg ctgaatcgcc atcgttacac cagtgattcg ttgccgccaa
     2701 catccagcga aacgttgctg gataagctga ttacccggct ggcggctagc ctgaaaagtc
     2761 cctttgcgct ggataaattt tgtgatgagg catcgtgcag tgagcgcgtt ttgcgtcagc
     2821 aatttcgcca gcagactgga atgaccatca atcaatatct gcgacaggtc agagtgtgtc
     2881 atgcgcaata tcttctccag catagccgcc tgttaatcag tgatatttcg accgaatgtg
     2941 gctttgaaga tagtaactat ttttcggtgg tgtttacccg ggaaaccggg atgacgccca
     3001 gccagtggcg tcatctcaat tcgcagaaag attaatcaca ctggctcacc ttcgggtggg
     3061 cctttctgcg tttatatact agagagagaa tataaaaagc cagattatta atccggcttt
     3121 tttattattt aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgaaaaa
     3181 aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat tttaacttgc
     3241 tatttctagc tctaaaaccc ggcaagctgc ccgtgcccgt gctcagtatc ttgttatccg
     3301 ctcacaatgt caattgttat ccgctcacaa ttaaaataaa aacagccgtg cctctttctg
     3361 gcaacggctg tttttattta ttcaattacg ccccgccctg ccactcatcg cagtactgtt
     3421 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     3481 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccata gtgaaaacgg
     3541 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     3601 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     3661 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     3721 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     3781 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac tccggatgag
     3841 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     3901 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     3961 taactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     4021 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
     4081 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     4141 gccaagccaa ataggccgt
//
