LOCUS       sequence-318471-        7095 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Rhamnose-inducible expression of 8xHIS-ABE8e in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7095)
  AUTHORS   Huang TP, Newby GA, Liu DR
  TITLE     Precision genome editing using cytosine and adenine base editors in 
            mammalian cells.
  JOURNAL   Nat Protoc. 2021 Feb;16(2):1089-1128. doi: 
            10.1038/s41596-020-00450-9. Epub 2021 Jan 18.
  PUBMED    33462442
REFERENCE   2  (bases 1 to 7095)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41596-020-00450-9"; journalName: "Nat Protoc"; date: 
            "2021-02"; volume: "16"; issue: "2"; pages: "1089-1128"
COMMENT     Sequence Label: pABE8e-protein
FEATURES             Location/Qualifiers
     source          1..7095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..91)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(100..130)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        195..313
                     /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             348..371
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             405..425
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1020..1043,1044..1046,1047..5120)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5166..5186
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5207..5225)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5221..5268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5366..5395
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     rep_origin      5425..5970
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     5871..5890
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6087..6104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(6235..6262)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     7009..7028
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta tttcattatg
       61 gtgaaagttg gaacctctta cgtgccgatc aagaagacgg tcaaaagcct ccggtcggag
      121 gcttttgact ttctgctatg gaggtcaggt atgatttaaa tggtcagtat tgagcgatat
      181 ctagagaatt cgtccaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc
      241 tggttgccaa tggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt
      301 tagactggtc gtaatgaaat tctttttaag aaggagatat acatatgcac catcaccatc
      361 atcatcatca caaacgtacc gcagatggta gcgaatttga aagcccgaaa aaaaagcgta
      421 aagttagcga agttgaattt agccacgaat actggatgcg tcatgcgttg acgctggcga
      481 agcgtgcgcg tgacgaacgc gaggtgccgg tgggtgctgt tctggtgctg aacaatcgtg
      541 taatcggtga aggttggaat cgcgcgatcg gtctgcatga tccaaccgcg cacgcagaaa
      601 tcatggcgct gcgtcagggt ggcctggtga tgcaaaatta ccgtctgatc gatgcgacgc
      661 tgtatgtgac cttcgaaccg tgtgtcatgt gtgccggtgc catgatccac agccgcattg
      721 gccgtgtggt ctttggcgtt cgtaacagca aacgtggcgc agcaggctct ctgatgaacg
      781 tcctgaatta cccgggtatg aaccatcgtg ttgagattac ggagggcatc ttagcggatg
      841 aatgtgctgc gctgctgtgc gatttctatc gcatgccgcg tcaagtcttt aacgcacaga
      901 agaaggcaca atctagcatt aactctggcg gttcgtctgg tggtagcagc ggcagcgaaa
      961 cgccgggtac cagcgagagc gcaaccccgg aaagctccgg tggcagctct ggcggctctg
     1021 acaaaaagta ttcgattggc ctggcgatcg gcacgaacag cgtcggttgg gctgttatta
     1081 ccgacgagta taaagtgccg agcaagaagt ttaaagtctt gggtaacacc gatcgccata
     1141 gcatcaaaaa gaatctgatt ggtgccctgc tgtttgatag cggcgaaacg gcagaagcga
     1201 cccgtctgaa acgtaccgca cgtcgtcgct acactcgtcg taaaaaccgc atttgctatt
     1261 tgcaagagat cttcagcaat gaaatggcca aagttgacga cagcttcttt caccgtctgg
     1321 aggagagctt tctggttgaa gaggataaga aacacgagcg tcacccgatc tttggtaata
     1381 tcgttgacga ggtcgcgtat cacgagaagt acccgacgat ctaccacttg cgtaagaaac
     1441 tggtggacag caccgataaa gccgatctgc gtctgattta cttggcgttg gcgcacatga
     1501 tcaagttccg cggtcatttc ctgatcgagg gtgacctgaa cccagataat tccgacgtcg
     1561 acaagctgtt catccagctg gttcagacct acaaccaatt gtttgaagaa aacccgatta
     1621 atgcatctgg tgtcgacgcg aaggcgattc tgagcgcgcg tctgagcaag tctcgtcgtc
     1681 tggagaatct gatcgcgcaa ttgccgggtg agaagaaaaa tggtttgttt ggcaacttga
     1741 tcgcgctgag cctgggtctg accccgaact ttaagagcaa tttcgacctg gcagaagatg
     1801 cgaaactgca gctgagcaaa gacacctatg atgacgacct ggataatctc ctggcacaaa
     1861 ttggtgacca gtatgcggac ctgttcctgg ctgcgaaaaa tctgtcggac gccattctgc
     1921 tgtcggacat tctgcgcgtt aacactgaga tcacgaaagc accgctgtct gcgagcatga
     1981 ttaaacgtta tgacgagcac catcaagacc tgaccctgct gaaggccctg gtccgtcagc
     2041 aactgccgga gaagtataaa gaaatttttt tcgatcagag caagaacggt tacgcaggct
     2101 atatcgatgg cggtgcgagc caagaagagt tttacaaatt cattaaaccg attttggaga
     2161 agatggacgg taccgaggag ttgctggtga aactgaatcg cgaagatctg ctgcgcaaac
     2221 aacgtacgtt cgataacggt agcatcccac accagattca cctgggtgag ctgcacgcga
     2281 ttctgcgtcg tcaggaagat ttctacccat tcctgaaaga taatcgtgaa aagattgaga
     2341 aaatcttgac gttccgcatt ccgtactacg tcggtccgct ggcacgtggc aacagccgtt
     2401 tcgcttggat gacgcgcaag agcgaggaaa ctatcacgcc gtggaacttt gaggaagtcg
     2461 tcgacaaagg tgcgagcgcg caaagcttta ttgagcgcat gaccaacttt gacaagaatc
     2521 tgccgaacga gaaagtcttg ccaaagcaca gcctgctgta cgagtatttc acggtgtaca
     2581 atgaactgac caaagttaag tatgtcaccg agggtatgcg taaaccagcg ttcctgtccg
     2641 gtgaacagaa gaaagcaatt gtggatctgt tgtttaagac gaaccgtaaa gtcacggtga
     2701 agcagttgaa agaggattac ttcaaaaaga tcgaatgctt tgacagcgtg gaaattagcg
     2761 gtgttgagga ccgtttcaac gccagcttgg gcacctacca tgatctgctg aaaatcatta
     2821 aggacaagga cttcctggat aacgaagaaa acgaggatat tctggaggat attgtcctga
     2881 cgctgaccct cttcgaagat cgcgaaatga ttgaagagag actgaaaact tacgcccact
     2941 tgtttgacga taaagtcatg aaacaactga agcgccgtcg ttacaccggt tggggtcgtc
     3001 tgagccgtaa gctgatcaac ggtatccgtg ataagcaatc tggcaaaacg atcctggact
     3061 ttctgaaaag cgatggtttc gcgaaccgca acttcatgca gcttattcac gacgattctc
     3121 tgacctttaa ggaggacatc caaaaagccc aggttagcgg tcagggcgat tccctgcatg
     3181 agcatattgc taacttagcg ggtagcccgg caatcaaaaa gggcattctg caaaccgtta
     3241 aggttgtgga cgagttggtt aaggtcatgg gccgtcataa gccggagaat atcgttattg
     3301 agatggcgcg cgagaatcag accacgcaga aaggccaaaa gaatagccgt gaacgtatga
     3361 aacgtattga agagggtatc aaagagctgg gcagccaaat cttgaaggaa catccggtgg
     3421 agaacacgca gctgcagaat gaaaagctgt acctgtacta tctgcaaaac ggtcgtgata
     3481 tgtacgttga ccaagagctg gacatcaacc gcctgagcga ttatgatgtt gatcacattg
     3541 tgccgcaatc ttttctgaag gacgacagca tcgacaacaa agttttgacc cgtagcgaca
     3601 agaatcgcgg taaaagcgat aatgttccga gcgaagaggt cgttaagaag atgaaaaact
     3661 attggcgtca gttgctgaat gcaaaactga ttacccaacg caaattcgac aatttgacta
     3721 aagcggagcg tggtggtctg agcgagctgg acaaggcggg tttcattaag cgtcaactgg
     3781 ttgaaactcg tcagatcacg aagcacgtgg cgcagattct ggacagccgc atgaatacta
     3841 agtacgatga aaatgataaa ctcattcgtg aggtcaaggt cattaccctg aagtctaagc
     3901 tcgtcagcga ttttcgtaaa gacttccagt tctataaggt gcgcgaaatc aacaattacc
     3961 accacgccca tgatgcatat ctgaacgccg tggtgggtac ggctctgatc aagaaatatc
     4021 cgaaactgga gtccgagttt gtctatggtg actacaaagt gtatgacgtc cgcaaaatga
     4081 tcgccaaatc ggaacaagag attggcaagg caaccgcgaa gtactttttc tattccaaca
     4141 ttatgaactt tttcaagacc gagatcaccc tggcaaacgg tgagatccgt aaacgtccgc
     4201 tgatcgaaac taacggtgaa accggtgaaa tcgtgtggga caaaggtcgt gactttgcaa
     4261 ccgtgcgtaa agtgctgtcg atgccacagg tcaacatcgt taagaaaacc gaagttcaaa
     4321 ctggtggctt tagcaaggag agcattctgc ctaagcgcaa cagcgacaag ctgattgcac
     4381 gtaagaagga ctgggacccg aagaagtacg gcggctttga ttccccgacg gttgcgtata
     4441 gcgttctcgt tgtggcgaaa gtcgagaaag gcaagagcaa aaagctgaaa agcgttaaag
     4501 aattgctggg tatcacgatt atggagcgta gcagcttcga aaagaatcct attgatttct
     4561 tggaagcaaa aggttacaag gaggtgaaga aagacttgat catcaaactg ccgaagtatt
     4621 ccctgttcga gctggaaaac ggccgcaaac gtatgctggc cagcgctggt gaactgcaaa
     4681 agggcaacga actggctctg ccgagcaaat atgtgaattt cctgtacctg gccagccact
     4741 acgagaaact gaagggctcc ccggaagata acgagcagaa gcagctgttc gttgagcagc
     4801 acaagcatta cctggacgag attatcgagc agatcagcga gtttagcaaa cgcgtgattc
     4861 tggcggatgc gaatttggat aaagttctgt cggcctataa taaacaccgc gacaaaccga
     4921 ttcgcgagca ggcggagaac attatccact tgttcaccct gaccaatctg ggtgctccgg
     4981 ctgctttcaa atactttgat accacgattg atcgcaaaag atatacgtcc accaaagagg
     5041 tactggacgc gaccctgatc caccagagca tcaccggctt gtacgaaacg cgcatcgatc
     5101 tgtctcaact gggcggtgac agcggtggtt cgaaacgcac agccgatgga tcagaatttg
     5161 aatctccgaa aaagaaacgc aaggtgtaag gttagagcgg ccgccaccgc tgagcaataa
     5221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     5281 actatatccg ggtaacgaat tcaagcttga tatcattcag gacgagcctc agactccagc
     5341 gtaactggac tgcaatcaac tcactggctc accttcacgg gtgggccttt cttcggtaga
     5401 agtcttctta ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc
     5461 tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag gttctctgag ctaccaactc
     5521 tttgaaccga ggtaactggc ttggaggagc gcagtcacca aaacttgtcc tttcagttta
     5581 gccttaaccg gcgcatgact tcaagactaa ctcctctaaa tcaattacca gtggctgctg
     5641 ccagtggtgc ttttgcatgt ctttccgggt tggactcaag acgatagtta ccggataagg
     5701 cgcagcggtc ggactgaacg gggggttcgt gcatacagtc cagcttggag cgaactgcct
     5761 acccggaact gagtgtcagg cgtggaatga gacaaacgcg gccataacag cggaatgaca
     5821 ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg agccgccagg gggaaacgcc
     5881 tggtatcttt atagtcctgt cgggtttcgc caccactgat ttgagcgtca gatttcgtga
     5941 tgcttgtcag gggggcggag cctatggaaa aacggctttg ccgcggccct ctcacttccc
     6001 tgttaagtat cttcctggca tcttccagga aatctccgcc ccgttcgtaa gccatttccg
     6061 ctcgccgcag tcgaacgacc gagcgtagcg agtcagtgag cgaggaagcg gaatatatcc
     6121 tgtatcacat attctgctga cgcaccggtg cagccttttt tctcctgcca catgaagcac
     6181 ttcactgaca ccctcatcag tgccaacata gtaagccagt atacactccg ctagcgcaga
     6241 aaggcccacc cgaaggtgag ccaggtgatt acatttgggc cctcattaga aaaactcatc
     6301 gagcatcaaa tgaaattgca atttattcat atcaggatta tcaataccat atttttgaaa
     6361 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     6421 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     6481 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     6541 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     6601 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg
     6661 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgagtgca accggcgcag
     6721 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     6781 gaacgctgtt tttccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     6841 aaaatgcttg atggtcggaa gtggcataaa ttccgtcagc cagtttagtc tgaccatctc
     6901 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     6961 gggcttccca tacaagcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     7021 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctcg acgtttcccg
     7081 ttgaatattg ctcat
//
