LOCUS       sequence-317804-        5323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mEmerald reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5323)
  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 5323)
  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: pNK141e2
FEATURES             Location/Qualifiers
     source          1..5323
                     /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..796
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVN
                     FKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             86..796
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIKVN
                     FKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(113..141)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     736..756
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(869..886)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1039..1125
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1217..1244
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1288..1833
                     /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     1734..1753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1880..1899)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1905..2402)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2351..2373
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2365..2382
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2366..2382
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2398..2414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2398..2414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2411..2433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2422..2438
                     /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(2446..2452,2453..2470,2471..2476))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2525..3607)
                     /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     3569..3588
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3608..3685)
                     /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     3805..3827
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3960..3979
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4351..5166)
                     /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
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLMEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5074..5093
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5158..5177
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcagtaa aggagaagaa cttttcactg gagttgtccc
      121 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
      181 tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact
      241 acctgttcca tggccaacac ttgtcactac tctgacttat ggtgttcaat gctttgcaag
      301 atacccagat catatgaaac agcatgactt tttcaagagt gccatgcccg aaggttatgt
      361 acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa
      421 gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga
      481 tggaaacatt cttggacaca aattggaata caactataac tcacacaagg tatacatcac
      541 ggcagacaaa caaaagaatg gaatcaaagt taacttcaaa actagacaca acattgaaga
      601 tggaagcgtt caactagcag accattatca acaaaatact ccaattggcg atggccctgt
      661 ccttttacca gacaaccatt acctgtccac acaatctaag ctttcgaaag atcccaacga
      721 aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat
      781 ggatgaacta tacaaataag tcgaccggct gctaacaaag cccgcggccg ctgaagatcg
      841 atctcgacga gtgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg
      901 gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc
      961 cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtcaccc catgcgagag
     1021 tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     1081 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     1141 ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     1201 aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacagagc
     1261 gtcagacccc ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg
     1321 ctctgaaaac gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa
     1381 ctctttgaac cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt
     1441 ttagccttaa ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg
     1501 ctgccagtgg tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata
     1561 aggcgcagcg gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg
     1621 cctacccgga actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg
     1681 acaccggtaa accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac
     1741 gcctggtatc tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg
     1801 tgatgcttgt caggggggcg gagcctatgg aaaaacggct ttgccgcggc cctctcggat
     1861 ctgtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     1921 tccgctatcg ctacgtgact gcctcgacct gcagcaattc caacgccatc aaaaataatt
     1981 cgcgtctggc cttcctgtag ccagctttca tcaacattaa atgtgagcga gtaacaaccc
     2041 gtcggattct ccgtgggaac aaacggcgga ttgaccgtaa tgggataggt cacgttggtg
     2101 tagatgggcg catcgtaacc gtgcatctgc cagtttgagg ggacgacgac agtatcggcc
     2161 tcaggaagat cgcactccag ccagctttcc ggcaccgctt ctggtgccgg aaaccaggca
     2221 aagcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     2281 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     2341 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaatccgt aatcatggtc
     2401 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2461 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taacttacat taattgcgtt
     2521 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2581 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     2641 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     2701 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     2761 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     2821 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     2881 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     2941 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     3001 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     3061 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     3121 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     3181 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     3241 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     3301 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     3361 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     3421 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     3481 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     3541 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     3601 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     3661 aggttttgca ccattccatg gtgtcggaat tgctgcaggt cgagggggtc atggctgcgc
     3721 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3781 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3841 caccgaaacg cgcgaggcag gatggcgccc aacagtcccc cggccacggg gcctgccacc
     3901 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     3961 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     4021 atgcgtccgg cgtagaggat ccgtcgacct acaggggggg ggggcgctga ggtctgcctc
     4081 gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc agccagaaag
     4141 tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg attttgaact
     4201 tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tccttcaact
     4261 cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt caagtcagcg taatgctctg
     4321 ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa
     4381 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     4441 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     4501 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     4561 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg
     4621 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     4681 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     4741 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     4801 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4861 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     4921 cggaagaggc ataaattcca tcagccagtt tagtctgacc atctcatctg taacatcatt
     4981 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     5041 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     5101 atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg
     5161 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
     5221 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttccat
     5281 caaaaaaata ttgacaacat aaaaaacttt gtgttatact tgt
//
