LOCUS       sequence-353269-        6903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-9S [rpoA].
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  AUTHORS   Bernath-Levin K, Schmidberger J, Honkanen S, Gutmann B, Sun YK, 
            Pullakhandam A, Colas des Francs-Small C, Bond CS, Small I
  TITLE     Cofactor-independent RNA editing by a synthetic S-type PPR protein.
  JOURNAL   Synth Biol (Oxf). 2021 Dec 23;7(1):ysab034. doi: 
            10.1093/synbio/ysab034. eCollection 2021.
  PUBMED    35128071
REFERENCE   2  (bases 1 to 6903)
  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: "Synth Biol 
            (Oxf)."; date: "2021-12-23"; volume: "7(1):ysab034. doi"; pages: " 
            10.1093/synbio/ysab034. eCollection 2021"
COMMENT     Sequence Label: pET-GFP-9S [rpoA]
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     285..302
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    317..456
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(382..401)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     542..564
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(558..749)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1558..2640)
                     /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     2602..2621
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2641..2718)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2924..2943
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3027..3046
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3027..3045
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3046..3070
                     /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             3085..3107
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3120..3137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(3138..3140,3141..3143,3144..3854)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3138..3140,3141..3143,3144..3854)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3183..3204)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3444..3463)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3791..3812
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3858..3878
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4794..4811
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4864..4882)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4878..4925
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4962..5417
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5049..5068)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5259..5280
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5509..6324)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5509..6324)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6232..6251
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6316..6335
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6446..6903,1..131)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
       61 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      121 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      181 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      241 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      301 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
      361 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
      421 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
      481 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
      541 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
      601 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
      661 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
      721 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
      781 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
      841 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
      901 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
      961 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     1021 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     1081 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     1141 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     1201 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     1261 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     1321 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     1381 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     1441 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     1501 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     1561 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     1621 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     1681 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     1741 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     1801 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     1861 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     1921 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     1981 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     2041 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     2101 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     2161 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     2221 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     2281 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     2341 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     2401 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     2461 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     2521 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     2581 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     2641 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     2701 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     2761 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     2821 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     2881 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     2941 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     3001 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     3061 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat accatgaaac
     3121 atcaccatca ccatcacatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     3181 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     3241 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     3301 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     3361 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     3421 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     3481 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     3541 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     3601 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     3661 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     3721 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     3781 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     3841 acgagctgta caaggcggag aatctttatt ttcagggcgc catgggaaat tctgtggtgt
     3901 cgtggaacgc tatgatcagc gggtacgcgc aaaacggccg gatcgatgaa gcgcgagaac
     3961 tatttgataa gatgccggag agagacgttg tctcctggaa tgcgatgatt tcaggatatg
     4021 cgcagaacgg gaggattgat gaggctcggg agttatttga caaaatgcca gagcgggacg
     4081 tggtttcttg gactgccatg atctccgggt acgcacaaaa cggacgcatt gacgaagcgc
     4141 gggagctctt tgataaaatg cccgagcgca acgtcgtgtc ttggaatgcc atgatttcgg
     4201 ggtatgccca aaatggtcgc atcgacgagg cgcgagaatt attcgacaag atgccagaac
     4261 ggtcggtggt gtcctggacc gctatgatta gtggctacgc ccaaaacggg cgaatagatg
     4321 aagcccgtga actatttgac aagatgcctg aacgcaatgt cgtgtcgtgg aatgcaatga
     4381 tctcagggta tgcgcagaat ggacgaatag acgaagcgcg agagctgttc gataaaatgc
     4441 cggaacgatc ggtagtatca tggacggcaa tgatatcggg gtacgctcaa aatggtcgga
     4501 ttgatgaggc acgcgaactg ttcgacaaaa tgcctgagag ggatgtcgtc tcatggaacg
     4561 cgatgatctc cggatatgct caaaatggcc ggattgacga ggcgagagaa ctcttcgata
     4621 agatgcccga gcgggacgtc gtttcgtgga cagccatgat cagtggttac gcacaaaacg
     4681 ggcgcatcga tgaagctagg gaactgttcg acaagatgcc cgaaagggat gtagtctcga
     4741 acaacgccat gtcggctgga gcgtcgcaga atggctaagc ggccgcactc gagcaccacc
     4801 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     4861 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     4921 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     4981 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5041 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5101 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5161 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5221 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5281 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     5341 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5401 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5461 ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc
     5521 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     5581 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     5641 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     5701 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     5761 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     5821 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     5881 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     5941 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     6001 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     6061 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     6121 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     6181 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     6241 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     6301 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     6361 ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6421 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6481 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6541 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     6601 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6661 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6721 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6781 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6841 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6901 tcg
//
