LOCUS       sequence-317843-        5284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS E2-Crimson reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5284)
  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 5284)
  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: pNK141n2
FEATURES             Location/Qualifiers
     source          1..5284
                     /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(830..847)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1000..1086
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1178..1205
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1249..1794
                     /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     1695..1714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1841..1860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1866..2363)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2312..2334
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2326..2343
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2327..2343
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2359..2375)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2359..2375)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2372..2394)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2383..2399
                     /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(2407..2413,2414..2431,2432..2437))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2486..3568)
                     /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     3530..3549
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3569..3646)
                     /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     3766..3788
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3921..3940
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4312..5127)
                     /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(4312..5127)
                     /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     5035..5054
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5119..5138
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcgacag caccgagaat gtgattaaac cgttcatgcg
      121 cttcaaagtt cacatggagg gttcggttaa cggtcacgag ttcgagatcg aaggcgtagg
      181 cgagggcaag ccgtatgaag gcactcaaac cgcgaaactg caagtgacta aaggcggccc
      241 gctgcctttt gcgtgggaca tcctgagccc gcaatttttc tacggttcta aagcatatat
      301 caaacaccca gcggatatcc cggactattt aaagcagtct tttccggaag gtttcaagtg
      361 ggaacgcgta atgaattttg aagatggtgg tgtcgtgacc gtcactcagg actcctccct
      421 gcaggatggc accttgatct atcacgttaa atttattggt gtgaattttc catctgatgg
      481 cccggtgatg cagaaaaaga cgttgggttg ggagccgtct accgaacgca actatccgcg
      541 cgatggtgtg ctgaaaggcg aaaatcatat ggcgctgaaa ctgaaaggtg gcggccatta
      601 tctgtgtgaa tttaaaagca tttacatggc caagaaacct gtgaagctgc ctggctatca
      661 ctatgtggat tacaaactgg acatcacctc tcataatgaa gattatacgg tcgtagagca
      721 atatgagcgc gccgaggctc gtcatcatct gtttcagtaa gtcgaccggc tgctaacaaa
      781 gcccgcggcc gctgaagatc gatctcgacg agtgagagaa gattttcagc ctgatacaga
      841 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
      901 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
      961 tggggtcacc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     1021 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     1081 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     1141 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     1201 ctttttgcgt ttctacagag cgtcagaccc cttaataaga tgatcttctt gagatcgttt
     1261 tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc
     1321 gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc
     1381 accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc
     1441 taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact
     1501 caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac
     1561 agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa
     1621 cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg
     1681 agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac
     1741 tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc
     1801 tttgccgcgg ccctctcgga tctgtatggt gcactctcag tacaatctgc tctgatgccg
     1861 catagttaag ccagtataca ctccgctatc gctacgtgac tgcctcgacc tgcagcaatt
     1921 ccaacgccat caaaaataat tcgcgtctgg ccttcctgta gccagctttc atcaacatta
     1981 aatgtgagcg agtaacaacc cgtcggattc tccgtgggaa caaacggcgg attgaccgta
     2041 atgggatagg tcacgttggt gtagatgggc gcatcgtaac cgtgcatctg ccagtttgag
     2101 gggacgacga cagtatcggc ctcaggaaga tcgcactcca gccagctttc cggcaccgct
     2161 tctggtgccg gaaaccaggc aaagcgccat tcgccattca ggctgcgcaa ctgttgggaa
     2221 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     2281 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     2341 agtgaatccg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2401 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2461 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2521 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     2581 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     2641 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     2701 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     2761 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     2821 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     2881 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     2941 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     3001 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     3061 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     3121 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     3181 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     3241 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     3301 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     3361 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     3421 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     3481 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     3541 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     3601 gctatcatgc cataccgcga aaggttttgc accattccat ggtgtcggaa ttgctgcagg
     3661 tcgagggggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3721 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3781 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca ggatggcgcc caacagtccc
     3841 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     3901 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     3961 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tccgtcgacc tacagggggg
     4021 gggggcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag gcctgaatcg
     4081 ccccatcatc cagccagaaa gtgagggagc cacggttgat gagagctttg ttgtaggtgg
     4141 accagttggt gattttgaac ttttgctttg ccacggaacg gtctgcgttg tcgggaagat
     4201 gcgtgatctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg
     4261 tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa
     4321 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     4381 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     4441 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     4501 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     4561 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     4621 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     4681 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     4741 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     4801 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     4861 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     4921 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     4981 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     5041 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca
     5101 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     5161 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
     5221 agacacaacg tggctttcca tcaaaaaaat attgacaaca taaaaaactt tgtgttatac
     5281 ttgt
//
