LOCUS       sequence-330050-        5341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mMaroon1 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5341)
  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 5341)
  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: pNK141o2
FEATURES             Location/Qualifiers
     source          1..5341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    115..138
                     /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             200..931
                     /codon_start=1
                     /product="bright, rapidly maturing, monomeric far-red 
                     fluorescent protein (Bajar et al., 2016)"
                     /label=mMaroon1
                     /translation="VSKGEELIKENMHTKLYLTGTVNNHYFECTAEGEGKPYEGTQTNR
                     IKVVRGGPLPFAFDILAPCFMYGSKTFINHPPDIPDYFKQSFPEGFTWERTTVYEDGGT
                     LTATQDTSLQDGCLIYNVQVRGENFPSNGPVMQKKTLGWEASTETLYPADGSLEGRLDW
                     ALKLVGGGHLHCRLETTYRSKKPAKNLKMPGVYFIDRRLERIKEADNETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(1001..1018)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1171..1257
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1349..1376
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1420..1965
                     /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     1866..1885
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2012..2031)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(2037..2534)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2483..2505
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2497..2514
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2498..2514
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2530..2546)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2530..2546)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2543..2565)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2554..2570
                     /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(2578..2584,2585..2602,2603..2608))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2657..3739)
                     /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     3701..3720
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3740..3817)
                     /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     3937..3959
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4092..4111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4483..5298)
                     /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(4483..5298)
                     /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     5206..5225
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5290..5309
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
       61 tggctttcca tcaaaaaaat attgacaaca taaaaaactt tgtgttatac ttgtggaatt
      121 gtgagcggat aacaattcta tatctgttat tttttccaac cacagatcta tgagcaaagg
      181 tgaagaactg tttaccggcg tgagcaaggg cgaggagctg atcaaggaga acatgcacac
      241 caagctgtac ctgacaggca ccgtgaacaa ccactacttc gaatgcaccg ccgaagggga
      301 gggcaagccc tacgagggca cccagaccaa caggatcaag gtggtgcgcg gaggccccct
      361 gccgttcgca ttcgacatcc tggccccctg ctttatgtac gggagcaaga ccttcattaa
      421 ccatccacct gacattcccg attattttaa gcagtccttc cctgagggct tcacatggga
      481 gagaaccacc gtttacgaag acgggggcac actcactgct acacaggaca ccagcctcca
      541 ggacggctgc ctgatctaca acgtccaagt cagaggggag aacttcccat ccaacggccc
      601 tgtgatgcag aagaaaacac tcggctggga ggccagtacc gagaccctgt accccgctga
      661 cggcagcctg gaaggcagac tggactgggc cctgaagctc gtgggcgggg gccacctgca
      721 ctgcagactg gaaaccacat acagatccaa gaaacccgca aagaacctca agatgcccgg
      781 cgtctatttt attgacagga gactggaaag aatcaaggag gccgacaatg agacctacgt
      841 cgagcagcac gaggtggctg tggccagata ctgcgacctc cctagcaaac tggggcacaa
      901 acttaatggc atggacgagc tgtacaagta agtcgaccgg ctgctaacaa agcccgcggc
      961 cgctgaagat cgatctcgac gagtgagaga agattttcag cctgatacag attaaatcag
     1021 aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac
     1081 ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtcac
     1141 cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac
     1201 tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
     1261 ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg
     1321 ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg
     1381 tttctacaga gcgtcagacc ccttaataag atgatcttct tgagatcgtt ttggtctgcg
     1441 cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag ggcggttttt cgaaggttct
     1501 ctgagctacc aactctttga accgaggtaa ctggcttgga ggagcgcagt caccaaaact
     1561 tgtcctttca gtttagcctt aaccggcgca tgacttcaag actaactcct ctaaatcaat
     1621 taccagtggc tgctgccagt ggtgcttttg catgtctttc cgggttggac tcaagacgat
     1681 agttaccgga taaggcgcag cggtcggact gaacgggggg ttcgtgcata cagtccagct
     1741 tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg aatgagacaa acgcggccat
     1801 aacagcggaa tgacaccggt aaaccgaaag gcaggaacag gagagcgcac gagggagccg
     1861 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacca ctgatttgag
     1921 cgtcagattt cgtgatgctt gtcagggggg cggagcctat ggaaaaacgg ctttgccgcg
     1981 gccctctcgg atctgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     2041 gccagtatac actccgctat cgctacgtga ctgcctcgac ctgcagcaat tccaacgcca
     2101 tcaaaaataa ttcgcgtctg gccttcctgt agccagcttt catcaacatt aaatgtgagc
     2161 gagtaacaac ccgtcggatt ctccgtggga acaaacggcg gattgaccgt aatgggatag
     2221 gtcacgttgg tgtagatggg cgcatcgtaa ccgtgcatct gccagtttga ggggacgacg
     2281 acagtatcgg cctcaggaag atcgcactcc agccagcttt ccggcaccgc ttctggtgcc
     2341 ggaaaccagg caaagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2401 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     2461 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaatcc
     2521 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2581 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaacttac
     2641 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2701 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     2761 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2821 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2881 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     2941 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     3001 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     3061 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     3121 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3181 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3241 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     3301 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     3361 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     3421 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3481 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3541 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3601 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3661 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3721 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3781 ccataccgcg aaaggttttg caccattcca tggtgtcgga attgctgcag gtcgaggggg
     3841 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3901 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3961 tttcaccgtc atcaccgaaa cgcgcgaggc aggatggcgc ccaacagtcc cccggccacg
     4021 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     4081 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     4141 atgccggcca cgatgcgtcc ggcgtagagg atccgtcgac ctacaggggg ggggggcgct
     4201 gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc gccccatcat
     4261 ccagccagaa agtgagggag ccacggttga tgagagcttt gttgtaggtg gaccagttgg
     4321 tgattttgaa cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct
     4381 gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag
     4441 cgtaatgctc tgccagtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     4501 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     4561 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     4621 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     4681 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     4741 caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     4801 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     4861 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     4921 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     4981 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     5041 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     5101 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     5161 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     5221 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgagc aagacgtttc
     5281 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     5341 t
//
