LOCUS       sequence-317798-        5326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS Sirius reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5326)
  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 5326)
  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: pNK141a2
FEATURES             Location/Qualifiers
     source          1..5326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(183..202)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             342..593
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKAHFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSVQSKLSKDPNEKRDHMVLLESVTAAGITLGMDELYK"
     primer_bind     complement(666..683)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      836..922
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1014..1041
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1085..1630
                     /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     1531..1550
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1677..1696)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1702..2199)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2148..2170
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2162..2179
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2163..2179
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2195..2211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2195..2211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2208..2230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2219..2235
                     /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(2243..2249,2250..2267,2268..2273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2322..3404)
                     /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     3366..3385
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3405..3482)
                     /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     3602..3624
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3757..3776
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4148..4963)
                     /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(4148..4963)
                     /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     4871..4890
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4955..4974
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5121..5144
                     /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)."
     primer_bind     complement(5248..5269)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 agctgaccct gaagctcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
       61 tgaccaccct gcaattcggc gtgctgtgct tcgcccgcta ccccgaccac atgaagcagc
      121 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgtacc atcttcttca
      181 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
      241 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
      301 tggagtacaa cgggataagc tcaaacgtat atatcaccgc cgacaagcag aagaacggca
      361 tcaaggccca cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc
      421 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
      481 tgagcgtcca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
      541 tggagtccgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagtcg
      601 accggctgct aacaaagccc gcggccgctg aagatcgatc tcgacgagtg agagaagatt
      661 ttcagcctga tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
      721 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
      781 agcgccgatg gtagtgtggg gtcaccccat gcgagagtag ggaactgcca ggcatcaaat
      841 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      901 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
      961 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
     1021 catcctgacg gatggccttt ttgcgtttct acagagcgtc agacccctta ataagatgat
     1081 cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct
     1141 tgcagggcgg tttttcgaag gttctctgag ctaccaactc tttgaaccga ggtaactggc
     1201 ttggaggagc gcagtcacca aaacttgtcc tttcagttta gccttaaccg gcgcatgact
     1261 tcaagactaa ctcctctaaa tcaattacca gtggctgctg ccagtggtgc ttttgcatgt
     1321 ctttccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc ggactgaacg
     1381 gggggttcgt gcatacagtc cagcttggag cgaactgcct acccggaact gagtgtcagg
     1441 cgtggaatga gacaaacgcg gccataacag cggaatgaca ccggtaaacc gaaaggcagg
     1501 aacaggagag cgcacgaggg agccgccagg gggaaacgcc tggtatcttt atagtcctgt
     1561 cgggtttcgc caccactgat ttgagcgtca gatttcgtga tgcttgtcag gggggcggag
     1621 cctatggaaa aacggctttg ccgcggccct ctcggatctg tatggtgcac tctcagtaca
     1681 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactgcc
     1741 tcgacctgca gcaattccaa cgccatcaaa aataattcgc gtctggcctt cctgtagcca
     1801 gctttcatca acattaaatg tgagcgagta acaacccgtc ggattctccg tgggaacaaa
     1861 cggcggattg accgtaatgg gataggtcac gttggtgtag atgggcgcat cgtaaccgtg
     1921 catctgccag tttgagggga cgacgacagt atcggcctca ggaagatcgc actccagcca
     1981 gctttccggc accgcttctg gtgccggaaa ccaggcaaag cgccattcgc cattcaggct
     2041 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     2101 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     2161 ttgtaaaacg acggccagtg aatccgtaat catggtcata gctgtttcct gtgtgaaatt
     2221 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2281 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     2341 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2401 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     2461 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     2521 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     2581 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     2641 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     2701 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     2761 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     2821 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     2881 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     2941 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     3001 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     3061 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     3121 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     3181 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     3241 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     3301 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     3361 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     3421 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca ttccatggtg
     3481 tcggaattgc tgcaggtcga gggggtcatg gctgcgcccc gacacccgcc aacacccgct
     3541 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3601 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcaggat
     3661 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     3721 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     3781 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatccg
     3841 tcgacctaca gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca
     3901 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga
     3961 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct
     4021 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa
     4081 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca
     4141 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     4201 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     4261 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     4321 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     4381 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     4441 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     4501 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     4561 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     4621 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     4681 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     4741 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     4801 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     4861 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     4921 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     4981 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     5041 aacatcagag attttgagac acaacgtggc tttccatcaa aaaaatattg acaacataaa
     5101 aaactttgtg ttatacttgt ggaattgtga gcggataaca attctatatc tgttattttt
     5161 tccaaccaca gatctatgag caaaggtgaa gaactgttta ccggcgtgag caagggcgag
     5221 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     5281 aggttcagcg tgtccggcga gggcgagggc gatgccacct acggca
//
