LOCUS       sequence-317841-        5308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS LSS-mKate2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5308)
  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 5308)
  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: pNK141l2
FEATURES             Location/Qualifiers
     source          1..5308
                     /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)."
     primer_bind     complement(854..871)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1024..1110
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1202..1229
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1273..1818
                     /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     1719..1738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1865..1884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1890..2387)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2336..2358
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2350..2367
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2351..2367
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2383..2399)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2383..2399)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2396..2418)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2407..2423
                     /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(2431..2437,2438..2455,2456..2461))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2510..3592)
                     /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     3554..3573
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3593..3670)
                     /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     3790..3812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3945..3964
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4336..5151)
                     /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(4336..5151)
                     /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     5059..5078
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5143..5162
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcagcga gctgattaag gagaacatgc acatgaagct
      121 gtacatggaa ggcaccgtga acaaccacca cttcaagtgc acatccgagg gcgaaggcaa
      181 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctaccctt
      241 cgccttcgac atcttggcta ccagcttcat gtacggcagc tacaccttca tcaaccacac
      301 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagagt
      361 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
      421 ttgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
      481 gcagaagaaa acactcggct gggaggccgg caccgagatg ctgtaccccg ctgacggcgg
      541 cctggaaggc agatctgacg acgccctgaa gctcgtgggc gggggccacc tgatctgcaa
      601 cttgaagagc acatacagat ccaagaaacc cgctaagaat ctcaaggtgc ccggcgtcta
      661 ctatgtggac cgaagactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
      721 gcacgaggtg gctgtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
      781 ttaagtcgac cggctgctaa caaagcccgc ggccgctgaa gatcgatctc gacgagtgag
      841 agaagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga
      901 atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga
      961 aacgccgtag cgccgatggt agtgtggggt caccccatgc gagagtaggg aactgccagg
     1021 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     1081 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag
     1141 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag
     1201 cagaaggcca tcctgacgga tggccttttt gcgtttctac agagcgtcag accccttaat
     1261 aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa
     1321 aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg
     1381 taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc
     1441 gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt
     1501 ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1561 actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga
     1621 gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga
     1681 aaggcaggaa caggagagcg cacgagggag ccgccagggg gaaacgcctg gtatctttat
     1741 agtcctgtcg ggtttcgcca ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg
     1801 gggcggagcc tatggaaaaa cggctttgcc gcggccctct cggatctgta tggtgcactc
     1861 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     1921 tgactgcctc gacctgcagc aattccaacg ccatcaaaaa taattcgcgt ctggccttcc
     1981 tgtagccagc tttcatcaac attaaatgtg agcgagtaac aacccgtcgg attctccgtg
     2041 ggaacaaacg gcggattgac cgtaatggga taggtcacgt tggtgtagat gggcgcatcg
     2101 taaccgtgca tctgccagtt tgaggggacg acgacagtat cggcctcagg aagatcgcac
     2161 tccagccagc tttccggcac cgcttctggt gccggaaacc aggcaaagcg ccattcgcca
     2221 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     2281 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     2341 tcacgacgtt gtaaaacgac ggccagtgaa tccgtaatca tggtcatagc tgtttcctgt
     2401 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2461 agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     2521 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2581 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     2641 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     2701 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     2761 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     2821 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     2881 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     2941 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     3001 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     3061 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     3121 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     3181 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     3241 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     3301 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     3361 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     3421 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     3481 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     3541 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     3601 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     3661 ccatggtgtc ggaattgctg caggtcgagg gggtcatggc tgcgccccga cacccgccaa
     3721 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3781 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3841 ggcaggatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     3901 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     3961 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     4021 aggatccgtc gacctacagg gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg
     4081 ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg gagccacggt
     4141 tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc tttgccacgg
     4201 aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca aaagttcgat
     4261 ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt gttacaacca
     4321 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     4381 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     4441 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     4501 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     4561 accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac
     4621 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     4681 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     4741 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     4801 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     4861 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     4921 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     4981 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5041 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     5101 ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca taacacccct
     5161 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg
     5221 tgcaatgtaa catcagagat tttgagacac aacgtggctt tccatcaaaa aaatattgac
     5281 aacataaaaa actttgtgtt atacttgt
//
