LOCUS       sequence-317839-        5302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS Katushka-9-5 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5302)
  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 5302)
  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: pNK141j2
FEATURES             Location/Qualifiers
     source          1..5302
                     /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(848..865)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1018..1104
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1196..1223
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1267..1812
                     /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     1713..1732
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1859..1878)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1884..2381)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2330..2352
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2344..2361
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2345..2361
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2377..2393)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2377..2393)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2390..2412)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2401..2417
                     /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(2425..2431,2432..2449,2450..2455))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2504..3586)
                     /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     3548..3567
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3587..3664)
                     /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     3784..3806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3939..3958
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4330..5145)
                     /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(4330..5145)
                     /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     5053..5072
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5137..5156
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggctctga gctgattagc gagaacatgc acatgaagct
      121 gtacatggag ggcaccgtga acgaccacca cttcaagtgc acatccgagg gcgaaggcaa
      181 gccctacgag ggcacccaga ccatgaagat caaggtggtc gagggcggcc ctttaccctt
      241 cgccttcgac atcctggcta ccagcttcat gtacggcagc aagaccttca tcaaccacac
      301 ccagggcatc cccgacttct ttaagcagtc cttccctgag ggcttcacat gggagagaat
      361 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tgcagaacgg
      421 ctgcctgatc tacaacgtca agatcaacgg ggtgaacttc ccatccaacg gccctgtgat
      481 gcagaagaaa acactgggct gggaggccag caccgagatg ctgtaccccg ctgacagcgg
      541 cctgaggggc cacagccaga tggccctgaa gttagtgggc gggggctacc tgcactgcag
      601 cttgaagacc acatacagat ccaagaaacc cgctaagaac ttgaagatgc ccggcttcta
      661 cttcgtggac aggaaactgg aaagaatcaa ggaggccgac aaagagacct acgtcgagca
      721 gcacgagatg gctgtggcca ggtactgcga cctgcctagc aaactggggc acagctaagt
      781 cgaccggctg ctaacaaagc ccgcggccgc tgaagatcga tctcgacgag tgagagaaga
      841 ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc
      901 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
      961 gtagcgccga tggtagtgtg gggtcacccc atgcgagagt agggaactgc caggcatcaa
     1021 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     1081 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
     1141 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag
     1201 gccatcctga cggatggcct ttttgcgttt ctacagagcg tcagacccct taataagatg
     1261 atcttcttga gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc
     1321 cttgcagggc ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg
     1381 gcttggagga gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga
     1441 cttcaagact aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat
     1501 gtctttccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa
     1561 cggggggttc gtgcatacag tccagcttgg agcgaactgc ctacccggaa ctgagtgtca
     1621 ggcgtggaat gagacaaacg cggccataac agcggaatga caccggtaaa ccgaaaggca
     1681 ggaacaggag agcgcacgag ggagccgcca gggggaaacg cctggtatct ttatagtcct
     1741 gtcgggtttc gccaccactg atttgagcgt cagatttcgt gatgcttgtc aggggggcgg
     1801 agcctatgga aaaacggctt tgccgcggcc ctctcggatc tgtatggtgc actctcagta
     1861 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     1921 cctcgacctg cagcaattcc aacgccatca aaaataattc gcgtctggcc ttcctgtagc
     1981 cagctttcat caacattaaa tgtgagcgag taacaacccg tcggattctc cgtgggaaca
     2041 aacggcggat tgaccgtaat gggataggtc acgttggtgt agatgggcgc atcgtaaccg
     2101 tgcatctgcc agtttgaggg gacgacgaca gtatcggcct caggaagatc gcactccagc
     2161 cagctttccg gcaccgcttc tggtgccgga aaccaggcaa agcgccattc gccattcagg
     2221 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     2281 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     2341 cgttgtaaaa cgacggccag tgaatccgta atcatggtca tagctgtttc ctgtgtgaaa
     2401 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     2461 gggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     2521 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     2581 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     2641 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     2701 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     2761 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     2821 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     2881 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     2941 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     3001 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     3061 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     3121 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     3181 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     3241 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     3301 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     3361 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     3421 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     3481 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     3541 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     3601 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattccatgg
     3661 tgtcggaatt gctgcaggtc gagggggtca tggctgcgcc ccgacacccg ccaacacccg
     3721 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3781 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagg
     3841 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     3901 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     3961 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     4021 cgtcgaccta cagggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact
     4081 cataccaggc ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga
     4141 gagctttgtt gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
     4201 ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc
     4261 aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
     4321 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     4381 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     4441 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     4501 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     4561 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     4621 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4681 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     4741 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     4801 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     4861 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     4921 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     4981 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     5041 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     5101 taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     5161 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     5221 gtaacatcag agattttgag acacaacgtg gctttccatc aaaaaaatat tgacaacata
     5281 aaaaactttg tgttatactt gt
//
