LOCUS       sequence-317800-        5323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mCerulean 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: pNK141c2
FEATURES             Location/Qualifiers
     source          1..5323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /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     1206..1228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1361..1380
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1752..2567)
                     /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(1752..2567)
                     /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     2475..2494
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2559..2578
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    2725..2748
                     /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)."
     CDS             2810..3520
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2837..2865)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3460..3480
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3593..3610)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3763..3849
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3941..3968
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4012..4557
                     /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     4458..4477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4604..4623)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4629..5126)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     5075..5097
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5089..5106
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5090..5106
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5122..5138)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5122..5138)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5135..5157)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5146..5162
                     /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(5170..5176,5177..5194,5195..5200))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(join(5249..5323,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattccat
     1081 ggtgtcggaa ttgctgcagg tcgagggggt catggctgcg ccccgacacc cgccaacacc
     1141 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     1201 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     1261 ggatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     1321 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     1381 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     1441 tccgtcgacc tacagggggg gggggcgctg aggtctgcct cgtgaagaag gtgttgctga
     1501 ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat
     1561 gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg ccacggaacg
     1621 gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag ttcgatttat
     1681 tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaatta
     1741 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     1801 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     1861 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     1921 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     1981 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     2041 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     2101 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     2161 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     2221 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     2281 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     2341 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     2401 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     2461 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     2521 tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcataac accccttgta
     2581 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     2641 atgtaacatc agagattttg agacacaacg tggctttcca tcaaaaaaat attgacaaca
     2701 taaaaaactt tgtgttatac ttgtggaatt gtgagcggat aacaattcta tatctgttat
     2761 tttttccaac cacagatcta tgagcaaagg tgaagaactg tttaccggca gtaaaggaga
     2821 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     2881 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
     2941 atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactctcac
     3001 ttggggtgtt caatgctttg caagataccc agatcatatg aaacagcatg actttttcaa
     3061 gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
     3121 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt
     3181 aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacgc
     3241 tatctcagac aatgtataca tcacggcaga caaacaaaag aatggaatca aagctaactt
     3301 caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
     3361 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc
     3421 taagctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
     3481 agctgctggg attacacatg gcatggatga actatacaaa taagtcgacc ggctgctaac
     3541 aaagcccgcg gccgctgaag atcgatctcg acgagtgaga gaagattttc agcctgatac
     3601 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     3661 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     3721 gtgtggggtc accccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     3781 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     3841 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     3901 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     3961 ggcctttttg cgtttctaca gagcgtcaga ccccttaata agatgatctt cttgagatcg
     4021 ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt
     4081 ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca
     4141 gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc
     4201 ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg
     4261 actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca
     4321 tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac
     4381 aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc
     4441 acgagggagc cgccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4501 cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac
     4561 ggctttgccg cggccctctc ggatctgtat ggtgcactct cagtacaatc tgctctgatg
     4621 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgcctcg acctgcagca
     4681 attccaacgc catcaaaaat aattcgcgtc tggccttcct gtagccagct ttcatcaaca
     4741 ttaaatgtga gcgagtaaca acccgtcgga ttctccgtgg gaacaaacgg cggattgacc
     4801 gtaatgggat aggtcacgtt ggtgtagatg ggcgcatcgt aaccgtgcat ctgccagttt
     4861 gaggggacga cgacagtatc ggcctcagga agatcgcact ccagccagct ttccggcacc
     4921 gcttctggtg ccggaaacca ggcaaagcgc cattcgccat tcaggctgcg caactgttgg
     4981 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     5041 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     5101 gccagtgaat ccgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5161 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     5221 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5281 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggc
//
