LOCUS       sequence-375887-        7684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Test the U-to-C editing activity of KP6:E67A on AtrpoA editing site,
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7684)
  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 7684)
  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-KP6-E67A_rpoA-U
FEATURES             Location/Qualifiers
     source          1..7684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      8..463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(95..114)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     305..326
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        489..593
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(594..662,663..1454)
                     /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(812..831)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1625..2213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2114..2133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2367..2384
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2399..2538
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2464..2483)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2624..2646
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2640..2831)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3640..4722)
                     /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     4684..4703
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4723..4800)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5006..5025
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5109..5128
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5109..5127
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5128..5152
                     /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             5167..5189
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5197..5229
                     /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"
     CDS             5245..5568
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEI
                     ADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLD
                     ANLA"
     CDS             5590..5607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5617..5637
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             7524..7541
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7594..7612)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7608..7655
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
       61 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
      121 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
      181 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
      241 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
      301 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
      361 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
      421 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
      481 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      541 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      601 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      661 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      721 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      781 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      841 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
      901 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
      961 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1021 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1081 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     1141 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1201 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1261 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1321 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1381 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1441 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1501 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1561 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1621 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1681 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1741 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     1801 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1861 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     1921 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     1981 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2041 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2101 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2161 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2221 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2281 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2341 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2401 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     2461 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     2521 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     2581 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tatccgggag ctgcatgtgt
     2641 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     2701 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     2761 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     2821 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     2881 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     2941 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     3001 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     3061 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     3121 ctttacgaaa cacggaaacc gaaaaccatt catgttgttg ctcaggtcgc agacgttttg
     3181 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     3241 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     3301 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     3361 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     3421 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     3481 cctacgagtt gcatgataaa gaaaacagtc ataagtgcgg cgacgatagt catgccccgc
     3541 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     3601 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3661 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3721 cgtattgggc gccagggtgg tttttctttt caccagtgat acgggcaaca gctgattgcc
     3781 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     3841 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cctcggtatc
     3901 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     3961 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     4021 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     4081 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     4141 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     4201 cagatgctcc acgcccagtc gcgtaccgtc ctcatgggag aaaataatac tgttgatggg
     4261 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     4321 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     4381 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     4441 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     4501 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     4561 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     4621 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     4681 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     4741 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     4801 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     4861 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     4921 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     4981 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     5041 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     5101 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     5161 aataattttg tttaacttta agaaggagat atacatatgg ctagcatgac tggtggacag
     5221 caaatgggtc gcatggccgg agggagcgat aaaattattc acctgactga cgacagtttt
     5281 gacacggatg tactcaaagc ggacggggcg atcctcgtcg atttctgggc agagtggtgc
     5341 ggtccgtgca aaatgatcgc cccgattctg gatgaaatcg ctgacgaata tcagggcaaa
     5401 ctgaccgttg caaaactgaa catcgatcaa aaccctggca ctgcgccgaa atatggcatc
     5461 cgtggtatcc cgactctgct gctgttcaaa aacggtgaag tggcggcaac caaagtgggt
     5521 gcactgtcta aaggtcagtt gaaagagttc ctcgacgcta acctggccgg ttctggttct
     5581 ggccatatgc accatcatca tcatcattct tctggtgaga atctttattt tcagggcgcc
     5641 atgggtaatt ctgttgtcac atggaattcc atgattaggg gctacgctag aagcggccag
     5701 cccgaggaag ctctgagcct cttctcccag atgagaagga gcggcatcaa gcccgatagc
     5761 tataccttcc ccttcgtgct gaaggcttgc gcttctctga gcagcctcga ggaaggaaaa
     5821 caaatccacg cccacgtcat taaatccggc ttcgagtccg atgtctacgt gcagagctcc
     5881 ctcattgata tgtacgccaa gtgcggatct ctggaggacg ccagaaaagt gtttgacgag
     5941 atgcccgaga ggaatgtcgt gagctggaac gctatgatca gcggctatgc ccagaatgga
     6001 caatccgaag aagctctgga gctgtttaga gagatgcaga aagagggcat caagccttcc
     6061 gagtttacct tttgctccgt gctgaaggct tgcgcctctc tgggatctct ggagatgggc
     6121 aagcaaattc acggctatgt gatcaagtcc ggctttgagt ccatcgtgtt cgtgggaaac
     6181 gccctcattg acatgtatgc caagtgcggc tctctggagg atgctagaaa ggtctttgat
     6241 gagatgcccg aaaggaccgt tgtgagctgg accgccatga tctccggata cgcccagaac
     6301 ggacagagcg aggaggccct cgagctcttc agagagatgc agagggaggg cgtgaaaccc
     6361 gacgaagtga cactgccttc cgtgctctcc gcttgcgcca atctgggcgc tctggagcaa
     6421 ggcaaacaga tccatgccta cgtcatcaag aacggctttg aatccgatgt gtttgtgggc
     6481 tccgctctga tcgacatgta cgctaaatgc ggctccatcg aggaggctag gaaagtgttc
     6541 gatgaaatgc ccgagaaaga cgttgtggct tggaactcaa tgataaaagg ttatggaatg
     6601 aaccatgaag gcaaaatggc cgtccaatgt ttcgaagata tgcaaaaaca aggtgtaaag
     6661 ccttccgccg tcacttttac ctgtttgctg accgcctgtt ctcacgccag cctcgtcagc
     6721 gaagggcagg agtattttaa aatgatgcga gaagagtacg ggatcgctcc gagggtggaa
     6781 cattacggct gcatggtcga tctgcttgct cggagtggcc atctgtatga agctgaaaag
     6841 ttcctggaga tgctctgtcc tcccaacgaa gggacatggg gggccctcct cagcgcttgt
     6901 aagacttatg gcgaggtgga gcttggtctg aggtgctttc agcagctcgt gcagcttaac
     6961 ccagaaagtg ccgcctggta cgtgcttatg gctgacatat acgctggcgc cgggcggtgg
     7021 gacgacgcat accggatcga agagctgagg aaacatgccg gagccaagaa gaaacctgcc
     7081 tccgcattga ttgaagtgaa caaaaaagtg cacgaatttg tggtcggcaa taatcagagc
     7141 gaggagatca gtgcgatgct taagtctctc aactctagaa tgaaggagga gggccatgta
     7201 cccaatctgg atctggttct caaacccgtg tcagatgagg agaaagaggc agcgctgtgt
     7261 gagcacgcag cgaagttggc aattgcattc ggcttgctga atacgccaca gggacagacc
     7321 ctgcgcgtta caaagaacct gagaatgtgc aatgactgcc acaatgcgtc aaagatcatc
     7381 tccaagattg agcgcagaga gattatcctg cgcgatgatt gctgcattca ccacttcaag
     7441 gacggactgt gctcctgcgg agacatgttc taactcgaga aatagaagga acatgtatta
     7501 cacgtgcaaa atttgagagc tagcaccacc accaccacca ctgagatccg gctgctaaca
     7561 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     7621 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     7681 tggc
//
