LOCUS       sequence-317801-        5323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS Sapphire 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: pNK141d2
FEATURES             Location/Qualifiers
     source          1..5323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(549..554,555..572,573..579)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    587..603
                     /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     592..614
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     611..627
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     611..627
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             623..1120
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     complement(643..660)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(643..659)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(652..674)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1126..1145
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(1192..1737)
                     /direction=LEFT
                     /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     complement(1272..1291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1781..1808)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1900..1986)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2139..2156
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2226..2939)
                     /codon_start=1
                     /product="Aequorea victoria green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2269..2289)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2884..2912
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     protein_bind    3001..3024
                     /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(3171..3190)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3182..3997
                     /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             3182..3997
                     /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     complement(3255..3274)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(4369..4388)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(4521..4543)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4663..4740
                     /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."
     CDS             join(4741..5323,1..500)
                     /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     complement(4760..4779)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
       61 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
      121 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
      181 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
      241 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
      301 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
      361 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
      421 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
      481 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
      541 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
      601 caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg tcgttttaca
      661 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
      721 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
      781 cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag
      841 ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat
      901 gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc
      961 gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag
     1021 ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaattg ctgcaggtcg
     1081 aggcagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     1141 ttgtactgag agtgcaccat acagatccga gagggccgcg gcaaagccgt ttttccatag
     1201 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc
     1261 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt
     1321 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac
     1381 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc
     1441 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa
     1501 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg
     1561 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca
     1621 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc
     1681 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa
     1741 gatcatctta ttaaggggtc tgacgctctg tagaaacgca aaaaggccat ccgtcaggat
     1801 ggccttctgc ttaatttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
     1861 gggccgttgc ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc
     1921 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     1981 tatttgatgc ctggcagttc cctactctcg catggggtga ccccacacta ccatcggcgc
     2041 tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc
     2101 caggcaaatt ctgttttatc agaccgcttc tgcgttctga tttaatctgt atcaggctga
     2161 aaatcttctc tcactcgtcg agatcgatct tcagcggccg cgggctttgt tagcagccgg
     2221 tcgacttatt tgtatagttc atccatgcca tgtgtaatcc cagcagctgt tacaaactca
     2281 agaaggacca tgtggtctct cttttcgttg ggatctttcg aaagggcaga ttgtatggac
     2341 aggtaatggt tgtctggtaa aaggacaggg ccatcgccaa ttggagtatt ttgttgataa
     2401 tggtctgcta gttgaacgct tccatcttca atgttgtgtc taattttgaa gttaactttg
     2461 attccattct tttgtttgtc tgccatgatg tatacattgt gtgagttaaa gttgtattcc
     2521 aatttgtgtc caagaatgtt tccatcttct ttaaaatcaa taccttttaa ctcgattcta
     2581 ttaacaaggg tatcaccttc aaacttgact tcagcacgtg tcttgtagtt cccgtcatct
     2641 ttgaaaaata tagttctttc ctgtacataa ccttcgggca tggcactctt gaaaaagtca
     2701 tgctgtttca tatgatctgg gtatctcgca aagcattgaa caccataaga gaaagtagtg
     2761 acaagtgttg gccatggaac aggtagtttt ccagtagtgc aaataaattt aagggtaagt
     2821 tttccgtatg ttgcatcacc ttcaccctct ccactgacag aaaatttgtg cccattaaca
     2881 tcaccatcta attcaacaag aattgggaca actccagtga aaagttcttc tcctttactg
     2941 ccggtaaaca gttcttcacc tttgctcata gatctgtggt tggaaaaaat aacagatata
     3001 gaattgttat ccgctcacaa ttccacaagt ataacacaaa gttttttatg ttgtcaatat
     3061 ttttttgatg gaaagccacg ttgtgtctca aaatctctga tgttacattg cacaagataa
     3121 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     3181 tatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga
     3241 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     3301 ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     3361 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     3421 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     3481 gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     3541 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     3601 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     3661 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     3721 agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     3781 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     3841 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     3901 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     3961 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     4021 acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact
     4081 tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca
     4141 aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc
     4201 tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct
     4261 tcttcacgag gcagacctca gcgccccccc cccctgtagg tcgacggatc ctctacgccg
     4321 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     4381 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg
     4441 tgggtatggt ggcaggcccc gtggccgggg gactgttggg cgccatcctg cctcgcgcgt
     4501 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4561 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4621 tgtcggggcg cagccatgac cccctcgacc tgcagcaatt ccgacaccat ggaatggtgc
     4681 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     4741 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     4801 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     4861 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     4921 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     4981 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     5041 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     5101 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     5161 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     5221 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     5281 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgc
//
