LOCUS       sequence-208441-        8186 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  W2.7-1 plasmid. Contains PR-sd5-BE2, PLac-sgRNA1 and is part of 
            CAMERA 2.7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8186)
  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 8186)
  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: pWT047c
FEATURES             Location/Qualifiers
     source          1..8186
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..951)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTVKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      999..1221
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      1716..1761
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1875..2561
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             join(2610..2633,2634..2636,2637..5123,5124..5126,
                     5127..6710)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6723..6977
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     misc_RNA        complement(7091..7166)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    7199..7215
                     /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    7222..7238
                     /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(7296..8087)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(8088..8159)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg gttcgttgtt tttgcgtgag
       61 ccatgagaac gaaccattga gatcatgctt actttgcatg tcactcaaaa attttgcctc
      121 aaaactggtg agctgaattt ttacagttaa agcatcgtgt agtgtttttc ttagtccgtt
      181 acgtaggtag gaatctgatg taatggttgt tggtattttg tcaccattca tttttatctg
      241 gttgttctca agttcggtta cgagatccat ttgtctatct agttcaactt ggaaaatcaa
      301 cgtatcagtc gggcggcctc gcttatcaac caccaatttc atattgctgt aagtgtttaa
      361 atctttactt attggtttca aaacccattg gttaagcctt ttaaactcat ggtagttatt
      421 ttcaagcatt aacatgaact taaattcatc aaggctaatc tctatatttg ccttgtgagt
      481 tttcttttgt gttagttctt ttaataacca ctcataaatc ctcatagagt atttgttttc
      541 aaaagactta acatgttcca gattatattt tatgaatttt tttaactgga aaagataagg
      601 caatatctct tcactaaaaa ctaattctaa tttttcgctt gagaacttgg catagtttgt
      661 ccactggaaa atctcaaagc ctttaaccaa aggattcctg atttccacag ttctcgtcat
      721 cagctctctg gttgctttag ctaatacacc ataagcattt tccctactga tgttcatcat
      781 ctgagcgtat tggttataag tgaacgatac cgtccgttct ttccttgtag ggttttcaat
      841 cgtggggttg agtagtgcca cacagcataa aattagcttg gtttcatgct ccgttaagtc
      901 atagcgacta atcgctagtt catttgcttt gaaaacaact aattcagaca tacatctcaa
      961 ttggtctagg tgattttaat cactatacca attgagatgg gctagtcaat gataattact
     1021 agtccttttc ctttgagttg tgggtatctg taaattctgc tagacctttg ctggaaaact
     1081 tgtaaattct gctagaccct ctgtaaattc cgctagacct ttgtgtgttt tttttgttta
     1141 tattcaagtg gttataattt atagaataaa gaaagaataa aaaaagataa aaagaataga
     1201 tcccagccct gtgtataact cactacttta gtcagttccg cagtattaca aaaggatgtc
     1261 gcaaacgctg tttgctcctc tacaaaacag accttaaaac cctaaaggct taagtagcac
     1321 cctcgcaagc tcgggcaaat cgctgaatat tccttttgtc tccgaccatc aggcacctga
     1381 gtcgctgtct ttttcgtgac attcagttcg ctgcgctcac ggctctggca gtgaatgggg
     1441 gtaaatggca ctacaggcgc cttttatgga ttcatgcaag gaaactaccc ataatacaag
     1501 aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg tctgctatgt ggtgctatct
     1561 gactttttgc tgttcagcag ttcctgccct ctgattttcc agtctgacca cttcggatta
     1621 tcccgtgaca ggtcattcag actggctaat gcacccagta aggcagcggt atcatcaaca
     1681 ggcttacccg tcttactgtc aagaggacat ccggtcaaat aaaacgaaag gctcagtcga
     1741 aagactgggc ctttcgtttt agtgccgtta attaagttat caccgcaagg gataaatatc
     1801 taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca cctgcaggtg
     1861 cagtaaggaa aaaaatgtct tctgaaaccg gtccggttgc ggttgacccg accctgcgtc
     1921 gtcgtatcga accgcacgaa ttcgaagttt tcttcgaccc gcgtgaactg cgtaaagaaa
     1981 cctgcctgct gtacgaaatc aactggggtg gtcgtcactc tatctggcgt cacacctctc
     2041 agaacaccaa caaacacgtt gaagttaact tcatcgaaaa attcaccacc gaacgttact
     2101 tctgcccgaa cacccgttgc tctatcacct ggttcctgtc ttggtctccg tgcggtgaat
     2161 gctctcgtgc gatcaccgaa ttcctgtctc gttacccgca cgttaccctg ttcatctaca
     2221 tcgcgcgtct gtaccaccac gcggacccgc gtaaccgtca gggtctgcgt gacctgatct
     2281 cttctggtgt taccatccag atcatgaccg aacaggaatc tggttactgc tggcgtaact
     2341 tcgttaacta ctctccgtct aacgaagcgc actggccgcg ttacccgcac ctgtgggttc
     2401 gtctgtacgt tctggaactg tattgcatca tcctgggtct gccgccgtgc ctgaacatcc
     2461 tgcgtcgtaa acagccgcag ctgaccttct tcaccatcgc gctgcagtct tgccactacc
     2521 agcgtctgcc gccgcacatc ctgtgggcga ccggtctgaa atccggtagc gaaacaccgg
     2581 ggacttcaga atcggccacc ccggagtctg ataagaaata ctcaataggc ttagctatcg
     2641 gcacaaatag cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt
     2701 tcaaggttct gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt
     2761 tatttgacag tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt
     2821 atacacgtcg gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga
     2881 aagtagatga tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga
     2941 agcatgaacg tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat
     3001 atccaactat ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc
     3061 gcttaatcta tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg
     3121 gagatttaaa tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct
     3181 acaatcaatt atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc
     3241 tttctgcacg attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg
     3301 agaagaaaaa tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt
     3361 ttaaatcaaa ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg
     3421 atgatgattt agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg
     3481 cagctaagaa tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa
     3541 taactaaggc tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact
     3601 tgactctttt aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt
     3661 ttgatcaatc aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat
     3721 tttataaatt tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga
     3781 aactaaatcg tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc
     3841 atcaaattca cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat
     3901 ttttaaaaga caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg
     3961 ttggtccatt ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa
     4021 caattacccc atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta
     4081 ttgaacgcat gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata
     4141 gtttgcttta tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg
     4201 aaggaatgcg aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac
     4261 tcttcaaaac aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa
     4321 tagaatgttt tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag
     4381 gtacctacca tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa
     4441 atgaagatat cttagaggat attgttttaa cattgacctt atttgaagat agggagatga
     4501 ttgaggaaag acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta
     4561 aacgtcgccg ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg
     4621 ataagcaatc tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca
     4681 attttatgca gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac
     4741 aagtgtctgg acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg
     4801 ctattaaaaa aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg
     4861 ggcggcataa gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa
     4921 agggccagaa aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag
     4981 gaagtcagat tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct
     5041 atctctatta tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc
     5101 gtttaagtga ttatgatgtc gatgccattg ttccacaaag tttccttaaa gacgattcaa
     5161 tagacaataa ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa
     5221 gtgaagaagt agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa
     5281 tcactcaacg taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg
     5341 ataaagctgg ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg
     5401 cacaaatttt ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag
     5461 aggttaaagt gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat
     5521 tctataaagt acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg
     5581 tcgttggaac tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg
     5641 attataaagt ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag
     5701 caaccgcaaa atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac
     5761 ttgcaaatgg agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa
     5821 ttgtctggga taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag
     5881 tcaatattgt caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac
     5941 caaaaagaaa ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg
     6001 gtggttttga tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag
     6061 ggaaatcgaa gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa
     6121 gttcctttga aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa
     6181 aagacttaat cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac
     6241 ggatgctggc tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat
     6301 atgtgaattt tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata
     6361 acgaacaaaa acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc
     6421 aaatcagtga attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta
     6481 gtgcatataa caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt
     6541 tatttacgtt gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg
     6601 atcgtaaacg atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca
     6661 tcactggtct ttatgaaaca cgcattgatt tgagtcagct aggaggtgac agcggcggtt
     6721 caatgacaaa tctgtcggat atcatcgaaa aagaaaccgg taaacagctg gttatccaag
     6781 aaagtatttt aatgcttccc gaagaagtcg aagaggtgat tggcaacaaa cccgaaagtg
     6841 atattctcgt gcatactgcg tatgatgagt cgaccgatga aaacgtcatg ctgctgacct
     6901 ccgacgcgcc cgagtataaa ccgtgggctt tggttatcca ggatagcaac ggtgaaaata
     6961 agattaaaat gttataaacg gagccaatgt acgcaaaaac cccgcttcag cggggttttt
     7021 tcgccaaaaa aaaccccgcc ctgtcagggg cggggttttt ttttatgttg aaaatctcct
     7081 tctaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     7141 taacttgcta tttctagctc taaaacccgg caagctgccc gtgcccgtgc tcagtatctt
     7201 gttatccgct cacaatgtca attgttatcc gctcacaatt aaatataatg accctcttga
     7261 taacccaaga gggcattttt taatgcccat ggcgtttatt tgccgactac cttggtgatc
     7321 tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct
     7381 tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc
     7441 aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg
     7501 ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc
     7561 ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc
     7621 ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt
     7681 gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg
     7741 tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg
     7801 atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg
     7861 gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt
     7921 acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg
     7981 gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact
     8041 acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact cttccttttt
     8101 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttaaatgt
     8161 atttagaaaa ataggccaaa tgccgt
//
