LOCUS       sequence-317842-        5305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS MiCy reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5305)
  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 5305)
  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: pNK141m2
FEATURES             Location/Qualifiers
     source          1..5305
                     /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..781
                     /codon_start=1
                     /product="dimeric Midoriishi-Cyan fluorescent protein"
                     /label=MiCy
                     /translation="VSYSKQGIAQEMRTKYRMEGSVNGHEFTIEGVGTGNPYEGKQMSE
                     LVIIKPKGKPLPFSFDILSTAFQYGNRCFTKYPADMPDYFKQAFPDGMSYERSFLFEDG
                     GVATASWSIRLEGNCFIHNSIYHGVNFPADGPVMKKQTIGWDKSFEKMSVAKEVLRGDV
                     TQFLLLEGGGYQRCRFHSTYKTEKPVAMPPSHVVEHQIVRTDLGQTAKGFKVKLEEHAE
                     AHVNPLKVK"
     primer_bind     complement(851..868)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1021..1107
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1199..1226
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1270..1815
                     /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     1716..1735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1862..1881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1887..2384)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2333..2355
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2347..2364
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2348..2364
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2380..2396)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2380..2396)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2393..2415)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2404..2420
                     /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(2428..2434,2435..2452,2453..2458))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2507..3589)
                     /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     3551..3570
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3590..3667)
                     /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     3787..3809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3942..3961
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4333..5148)
                     /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(4333..5148)
                     /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     5056..5075
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5140..5159
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcgtgtc ttattcaaag caaggcatcg cacaagaaat
      121 gcggacgaaa taccgtatgg aaggcagtgt caatggccat gagttcacga tcgaaggtgt
      181 aggaactgga aacccttacg aagggaaaca gatgtccgaa ttagtgatca tcaagcctaa
      241 gggaaaaccc cttccattct cctttgacat actgtcaaca gcctttcaat atggaaacag
      301 atgcttcaca aagtaccctg ccgacatgcc tgactatttc aagcaagcat tcccagatgg
      361 aatgtcatat gaaaggtcat ttctatttga ggatggagga gttgctacag ccagctggag
      421 cattcgtctc gaaggaaatt gcttcatcca caattccatc tatcatggcg taaactttcc
      481 cgctgatgga cccgtaatga agaagcagac aattggctgg gataagtcct tcgaaaaaat
      541 gagtgtggct aaagaggtgc taagaggtga tgtgactcag tttcttctgc tcgaaggagg
      601 tggttaccag agatgccggt ttcactccac ttacaaaacg gagaagccag tcgcaatgcc
      661 cccgagtcat gtcgtagaac atcaaattgt gaggaccgac cttggccaaa ctgcaaaagg
      721 cttcaaggtc aagctggaag aacatgctga ggctcatgtt aaccctttga aggttaaata
      781 agtcgaccgg ctgctaacaa agcccgcggc cgctgaagat cgatctcgac gagtgagaga
      841 agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt
      901 tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
      961 gccgtagcgc cgatggtagt gtggggtcac cccatgcgag agtagggaac tgccaggcat
     1021 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     1081 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa
     1141 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag
     1201 aaggccatcc tgacggatgg cctttttgcg tttctacaga gcgtcagacc ccttaataag
     1261 atgatcttct tgagatcgtt ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac
     1321 cgccttgcag ggcggttttt cgaaggttct ctgagctacc aactctttga accgaggtaa
     1381 ctggcttgga ggagcgcagt caccaaaact tgtcctttca gtttagcctt aaccggcgca
     1441 tgacttcaag actaactcct ctaaatcaat taccagtggc tgctgccagt ggtgcttttg
     1501 catgtctttc cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcggact
     1561 gaacgggggg ttcgtgcata cagtccagct tggagcgaac tgcctacccg gaactgagtg
     1621 tcaggcgtgg aatgagacaa acgcggccat aacagcggaa tgacaccggt aaaccgaaag
     1681 gcaggaacag gagagcgcac gagggagccg ccagggggaa acgcctggta tctttatagt
     1741 cctgtcgggt ttcgccacca ctgatttgag cgtcagattt cgtgatgctt gtcagggggg
     1801 cggagcctat ggaaaaacgg ctttgccgcg gccctctcgg atctgtatgg tgcactctca
     1861 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     1921 ctgcctcgac ctgcagcaat tccaacgcca tcaaaaataa ttcgcgtctg gccttcctgt
     1981 agccagcttt catcaacatt aaatgtgagc gagtaacaac ccgtcggatt ctccgtggga
     2041 acaaacggcg gattgaccgt aatgggatag gtcacgttgg tgtagatggg cgcatcgtaa
     2101 ccgtgcatct gccagtttga ggggacgacg acagtatcgg cctcaggaag atcgcactcc
     2161 agccagcttt ccggcaccgc ttctggtgcc ggaaaccagg caaagcgcca ttcgccattc
     2221 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     2281 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     2341 cgacgttgta aaacgacggc cagtgaatcc gtaatcatgg tcatagctgt ttcctgtgtg
     2401 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     2461 ctggggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     2521 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2581 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     2641 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     2701 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     2761 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     2821 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     2881 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     2941 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     3001 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     3061 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     3121 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     3181 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     3241 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     3301 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     3361 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3421 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     3481 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     3541 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     3601 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcca
     3661 tggtgtcgga attgctgcag gtcgaggggg tcatggctgc gccccgacac ccgccaacac
     3721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc
     3841 aggatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     3901 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     3961 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     4021 atccgtcgac ctacaggggg ggggggcgct gaggtctgcc tcgtgaagaa ggtgttgctg
     4081 actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag ccacggttga
     4141 tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt gccacggaac
     4201 ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa gttcgattta
     4261 ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt
     4321 aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     4381 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     4441 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     4501 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     4561 atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg
     4621 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     4681 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     4741 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     4801 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     4861 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     4921 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     4981 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     5041 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     5101 atttaatcgc ggcctcgagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     5161 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     5221 aatgtaacat cagagatttt gagacacaac gtggctttcc atcaaaaaaa tattgacaac
     5281 ataaaaaact ttgtgttata cttgt
//
