LOCUS       sequence-375932-        7690 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Test the U-to-C editing activity of KP4 on AtrpoA editing site, in 
            E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7690)
  AUTHORS   Ichinose M, Kawabata M, Akaiwa Y, Shimajiri Y, Nakamura I, Tamai T, 
            Nakamura T, Yagi Y, Gutmann B
  TITLE     U-to-C RNA editing by synthetic PPR-DYW proteins in bacteria and 
            human culture cells.
  JOURNAL   Commun Biol. 2022 Sep 15;5(1):968. doi: 10.1038/s42003-022-03927-3.
  PUBMED    36109586
REFERENCE   2  (bases 1 to 7690)
  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: "Commun 
            Biol."; date: "2022-09-15"; volume: "5(1):968. doi"; pages: " 
            10.1038/s42003-022-03927-3"
COMMENT     Sequence Label: pET-PLS-KP4_rpoA-U
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             12..335
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEI
                     ADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD
                     ANLA"
     CDS             357..374
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             384..404
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2297..2314
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2367..2385)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2381..2428
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2465..2920
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2552..2571)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2762..2783
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2946..3050
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3051..3119,3120..3911)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3269..3288)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4082..4670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4571..4590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4824..4841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4856..4995
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4921..4940)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5081..5103
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5097..5288)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6097..7179)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7141..7160
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7180..7257)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7463..7482
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7566..7585
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7566..7584
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7585..7609
                     /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)."
     RBS             7624..7646
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7654..7686
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
ORIGIN
        1 tggccggagg gagcgataaa attattcacc tgactgacga cagttttgac acggatgtac
       61 tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa
      121 tgatcgcccc gattctggat gaaatcgctg acgaatatca gggcaaactg accgttgcaa
      181 aactgaacat cgatcaaaac cctggcactg cgccgaaata tggcatccgt ggtatcccga
      241 ctctgctgct gttcaaaaac ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag
      301 gtcagttgaa agagttcctc gacgctaacc tggccggttc tggttctggc catatgcacc
      361 atcatcatca tcattcttct ggtgagaatc tttattttca gggcgccatg ggtaattctg
      421 ttgtcacatg gaattccatg attaggggct acgctagaag cggccagccc gaggaagctc
      481 tgagcctctt ctcccagatg agaaggagcg gcatcaagcc cgatagctat accttcccct
      541 tcgtgctgaa ggcttgcgct tctctgagca gcctcgagga aggaaaacaa atccacgccc
      601 acgtcattaa atccggcttc gagtccgatg tctacgtgca gagctccctc attgatatgt
      661 acgccaagtg cggatctctg gaggacgcca gaaaagtgtt tgacgagatg cccgagagga
      721 atgtcgtgag ctggaacgct atgatcagcg gctatgccca gaatggacaa tccgaagaag
      781 ctctggagct gtttagagag atgcagaaag agggcatcaa gccttccgag tttacctttt
      841 gctccgtgct gaaggcttgc gcctctctgg gatctctgga gatgggcaag caaattcacg
      901 gctatgtgat caagtccggc tttgagtcca tcgtgttcgt gggaaacgcc ctcattgaca
      961 tgtatgccaa gtgcggctct ctggaggatg ctagaaaggt ctttgatgag atgcccgaaa
     1021 ggaccgttgt gagctggacc gccatgatct ccggatacgc ccagaacgga cagagcgagg
     1081 aggccctcga gctcttcaga gagatgcaga gggagggcgt gaaacccgac gaagtgacac
     1141 tgccttccgt gctctccgct tgcgccaatc tgggcgctct ggagcaaggc aaacagatcc
     1201 atgcctacgt catcaagaac ggctttgaat ccgatgtgtt tgtgggctcc gctctgatcg
     1261 acatgtacgc taaatgcggc tccatcgagg aggctaggaa agtgttcgat gaaatgcccg
     1321 agaaagacgt tgtctcttgg aatgcaatga ttgccggtta tgcccaatgc gggaatattg
     1381 aattggctcg gcaatgtttc gaagatatgc aacaggaagg catcaaacca agcgaggtga
     1441 ctttcacctc aatacttgct gcctgctctc atgccggtct ggtcgacgaa ggctgtaaat
     1501 acttcaagtc catgaaagag gatcacggga taataccaag gacacaccac tacggctgta
     1561 tggttgacct tctgggccgc gccggttacc ttaatgaggc cgaaaatctc ctgcagacga
     1621 tccagcctaa catcattgga tgggggagcc ttttgactgc ttgtcgcacc tatgggaatg
     1681 tagagctggg ccgacgctgt ttcgatcaga tcgttcagtt ggacccccat agcgcatccg
     1741 gctatgtcct gatgtccggc atttatagcg acgctcagat gtgggaggat gccgcaaaaa
     1801 tagaggagct caggcagcat gccggggctt ggaagaagcc tgcaaaagca tggatcgaag
     1861 tgaacaacaa agtgcatgag tttatcgtgg gcgataaaag ccacccccag tccgaggaaa
     1921 tctatgccat gctcaagaag ctcagtaacc ggatgaagga agagggatac gtacccaaga
     1981 tcgacctggt gctgcagcag atgtctgacg aagataaaga ggatgcactc tgcgggcatt
     2041 gcgagaaact ggccatcgcg tttggactga tttctacccc tccgggaaca accattcggg
     2101 cgacaaagaa cctgagggtg tgcatggact gtcacaacgc tactaagatc atctcaaaga
     2161 tcgagaagag agaaattatt attagagatg cttacagact gcaccacttt aaggacggat
     2221 catgcagttg cggagactac tactaactcg agaaatagaa ggaacatgta ttacacgtgc
     2281 aaaatttgag agctagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     2341 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2401 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2461 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2521 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2581 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2641 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2701 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2761 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2821 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2881 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     2941 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3001 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3061 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3121 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3181 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3241 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3301 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3361 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3421 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3481 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3541 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     3601 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3661 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3721 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3781 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     3841 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3901 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     3961 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4021 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4081 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4141 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4201 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4261 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4321 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4381 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4441 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4501 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4561 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4621 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4681 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4741 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4801 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4861 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4921 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4981 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     5041 ctgctcccgg catccgctta cagacaagct gtgaccgtat ccgggagctg catgtgtcag
     5101 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     5161 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     5221 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     5281 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     5341 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     5401 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     5461 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     5521 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     5581 tacgaaacac ggaaaccgaa aaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5641 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5701 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5761 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5821 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5881 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     5941 acgagttgca tgataaagaa aacagtcata agtgcggcga cgatagtcat gccccgcgcc
     6001 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6061 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6121 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6181 attgggcgcc agggtggttt ttcttttcac cagtgatacg ggcaacagct gattgccctt
     6241 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     6301 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtcct cggtatcgtc
     6361 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     6421 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     6481 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     6541 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6601 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6661 atgctccacg cccagtcgcg taccgtcctc atgggagaaa ataatactgt tgatgggtgt
     6721 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6781 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6841 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6901 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6961 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     7021 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     7081 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     7141 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     7201 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     7261 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     7321 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7381 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     7441 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     7501 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     7561 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     7621 aattttgttt aactttaaga aggagatata catatggcta gcatgactgg tggacagcaa
     7681 atgggtcgca
//
