LOCUS       sequence-317837-        5317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mCherry reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5317)
  AUTHORS   Kolber NS, Fattal R, Bratulic S, Carver GD, Badran AH
  TITLE     Orthogonal translation enables heterologous ribosome engineering in 
            E. coli.
  JOURNAL   Nat Commun. 2021 Jan 26;12(1):599. doi: 10.1038/s41467-020-20759-z.
  PUBMED    33500394
REFERENCE   2  (bases 1 to 5317)
  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: "Nat 
            Commun."; date: "2021-01-26"; volume: "12(1):599. doi"; pages: " 
            10.1038/s41467-020-20759-z"
COMMENT     Sequence Label: pNK141i2
FEATURES             Location/Qualifiers
     source          1..5317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..24
                     /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             86..793
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(228..246)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     496..515
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     690..713
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(863..880)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1033..1119
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1211..1238
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1282..1827
                     /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     1728..1747
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1874..1893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1899..2396)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2345..2367
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2359..2376
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2360..2376
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2392..2408)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2392..2408)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2405..2427)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2416..2432
                     /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)."
     promoter        complement(join(2440..2446,2447..2464,2465..2470))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2519..3601)
                     /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     3563..3582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3602..3679)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     3799..3821
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3954..3973
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4345..5160)
                     /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(4345..5160)
                     /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     5068..5087
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5152..5171
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcgtgag caagggcgag gaggataaca tggccatcat
      121 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
      181 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      241 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
      301 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
      361 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
      421 ccaggactcc tccctacagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
      481 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
      541 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
      601 ggacggcggc cactacgacg ccgaggtcaa gaccacctac aaggccaaga agcccgtgca
      661 gctgcccggc gcctacaacg tcaacatcaa gctggacatc acctcccaca acgaggacta
      721 caccatcgtg gaacagtacg agcgcgccga gggccgccac tccaccggcg gcatggacga
      781 gctgtacaag taagtcgacc ggctgctaac aaagcccgcg gccgctgaag atcgatctcg
      841 acgagtgaga gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga
      901 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
      961 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc accccatgcg agagtaggga
     1021 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     1081 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     1141 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     1201 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca gagcgtcaga
     1261 ccccttaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct cttgctctga
     1321 aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta ccaactcttt
     1381 gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt cagtttagcc
     1441 ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg gctgctgcca
     1501 gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg gataaggcgc
     1561 agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga actgcctacc
     1621 cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg aatgacaccg
     1681 gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg aaacgcctgg
     1741 tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat ttcgtgatgc
     1801 ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc ggatctgtat
     1861 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     1921 atcgctacgt gactgcctcg acctgcagca attccaacgc catcaaaaat aattcgcgtc
     1981 tggccttcct gtagccagct ttcatcaaca ttaaatgtga gcgagtaaca acccgtcgga
     2041 ttctccgtgg gaacaaacgg cggattgacc gtaatgggat aggtcacgtt ggtgtagatg
     2101 ggcgcatcgt aaccgtgcat ctgccagttt gaggggacga cgacagtatc ggcctcagga
     2161 agatcgcact ccagccagct ttccggcacc gcttctggtg ccggaaacca ggcaaagcgc
     2221 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     2281 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     2341 ttttcccagt cacgacgttg taaaacgacg gccagtgaat ccgtaatcat ggtcatagct
     2401 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     2461 aaagtgtaaa gcctggggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     2521 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2581 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     2641 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     2701 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     2761 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     2821 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     2881 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     2941 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     3001 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     3061 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     3121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     3181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     3241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     3301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     3361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     3421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     3481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     3541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     3601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     3661 tgcaccattc catggtgtcg gaattgctgc aggtcgaggg ggtcatggct gcgccccgac
     3721 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3781 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3841 aacgcgcgag gcaggatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     3901 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     3961 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     4021 ccggcgtaga ggatccgtcg acctacaggg ggggggggcg ctgaggtctg cctcgtgaag
     4081 aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag aaagtgaggg
     4141 agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg aacttttgct
     4201 ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa
     4261 aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg
     4321 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     4381 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     4441 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     4501 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     4561 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     4621 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4681 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     4741 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4801 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4861 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4921 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4981 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     5041 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     5101 atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat
     5161 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     5221 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt ccatcaaaaa
     5281 aatattgaca acataaaaaa ctttgtgtta tacttgt
//
