LOCUS       sequence-317840-        5341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mCarmine 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: pNK141k2
FEATURES             Location/Qualifiers
     source          1..5341
                     /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..817
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein with 
                     favorable expression and pKa properties and with peak 
                     emission at 675 nm (Fabritius et al., 2018)"
                     /label=mCarmine
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQR
                     IKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LKVKQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTETLYPADGGLRGYNTM
                     ALKLVGGGHLQTFLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     primer_bind     complement(887..904)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1057..1143
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1235..1262
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1306..1851
                     /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     1752..1771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1898..1917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1923..2420)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2369..2391
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2383..2400
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2384..2400
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2416..2432)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2416..2432)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2429..2451)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2440..2456
                     /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(2464..2470,2471..2488,2489..2494))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2543..3625)
                     /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     3587..3606
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3626..3703)
                     /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     3823..3845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3978..3997
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4369..5184)
                     /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(4369..5184)
                     /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     5092..5111
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5176..5195
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcgtgtc taagggcgaa gagctgatta aggagaacat
      121 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga
      181 gggcgaaggc aagccctacg agggcaccca gacccagaga atcaaggtgg tcgagggcgg
      241 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcaagacctt
      301 catcaaccac acccagggca tccccgattt ctttaagcag tccttccctg agggcttcac
      361 atgggagaga gtcaccacat acgaagacgg gggcgtgctg aaggtgaaac aggacaccag
      421 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa
      481 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc aacaccgaga ccctgtaccc
      541 cgctgacggc ggcctgcgcg gctataacac gatggccctg aagctcgtgg gcgggggcca
      601 cctgcagacg ttcctcaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
      661 gcccggcgtc tactttgtgg accgcagact ggaaagaatc aaggaggccg acaaagagac
      721 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
      781 gcacaaactt aatggcatgg acgagctgta caagtaagtc gaccggctgc taacaaagcc
      841 cgcggccgct gaagatcgat ctcgacgagt gagagaagat tttcagcctg atacagatta
      901 aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg
      961 tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg
     1021 ggtcacccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
     1081 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     1141 aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga
     1201 cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt
     1261 tttgcgtttc tacagagcgt cagacccctt aataagatga tcttcttgag atcgttttgg
     1321 tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa
     1381 ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag cgcagtcacc
     1441 aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta actcctctaa
     1501 atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg ttggactcaa
     1561 gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt
     1621 ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg agacaaacgc
     1681 ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga gcgcacgagg
     1741 gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga
     1801 tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa aaacggcttt
     1861 gccgcggccc tctcggatct gtatggtgca ctctcagtac aatctgctct gatgccgcat
     1921 agttaagcca gtatacactc cgctatcgct acgtgactgc ctcgacctgc agcaattcca
     1981 acgccatcaa aaataattcg cgtctggcct tcctgtagcc agctttcatc aacattaaat
     2041 gtgagcgagt aacaacccgt cggattctcc gtgggaacaa acggcggatt gaccgtaatg
     2101 ggataggtca cgttggtgta gatgggcgca tcgtaaccgt gcatctgcca gtttgagggg
     2161 acgacgacag tatcggcctc aggaagatcg cactccagcc agctttccgg caccgcttct
     2221 ggtgccggaa accaggcaaa gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     2281 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     2341 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     2401 gaatccgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2461 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2521 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2581 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     2641 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     2701 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     2761 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     2821 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     2881 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     2941 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     3001 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     3061 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     3121 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     3181 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     3241 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     3301 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     3361 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     3421 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     3481 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     3541 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     3601 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     3661 atcatgccat accgcgaaag gttttgcacc attccatggt gtcggaattg ctgcaggtcg
     3721 agggggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3781 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3841 agaggttttc accgtcatca ccgaaacgcg cgaggcagga tggcgcccaa cagtcccccg
     3901 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     3961 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     4021 ccggtgatgc cggccacgat gcgtccggcg tagaggatcc gtcgacctac aggggggggg
     4081 ggcgctgagg tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc
     4141 catcatccag ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc
     4201 agttggtgat tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg
     4261 tgatctgatc cttcaactca gcaaaagttc gatttattca acaaagccgc cgtcccgtca
     4321 agtcagcgta atgctctgcc agtgttacaa ccaattaacc aattctgatt agaaaaactc
     4381 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     4441 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     4501 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     4561 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     4621 gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     4681 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     4741 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     4801 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     4861 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     4921 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     4981 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     5041 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     5101 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tcgagcaaga
     5161 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     5221 ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga
     5281 cacaacgtgg ctttccatca aaaaaatatt gacaacataa aaaactttgt gttatacttg
     5341 t
//
