LOCUS       sequence-317799-        5308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mTagBFP 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: pNK141b2
FEATURES             Location/Qualifiers
     source          1..5308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(118..135)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      288..374
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      466..493
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      537..1082
                     /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     983..1002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1129..1148)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1154..1651)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     1600..1622
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1614..1631
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1615..1631
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1647..1663)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1647..1663)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1660..1682)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1671..1687
                     /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(1695..1701,1702..1719,1720..1725))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(1774..2856)
                     /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     2818..2837
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2857..2934)
                     /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     3054..3076
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3209..3228
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(3600..4415)
                     /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(3600..4415)
                     /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     4323..4342
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4407..4426
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4573..4596
                     /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             join(4658..5308,1..48)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
ORIGIN
        1 gccagatact gcgacctccc tagcaaactg gggcacaagc ttaattaagt cgaccggctg
       61 ctaacaaagc ccgcggccgc tgaagatcga tctcgacgag tgagagaaga ttttcagcct
      121 gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag
      181 tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga
      241 tggtagtgtg gggtcacccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa
      301 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
      361 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
      421 ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga
      481 cggatggcct ttttgcgttt ctacagagcg tcagacccct taataagatg atcttcttga
      541 gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc
      601 ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga
      661 gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact
      721 aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg
      781 gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc
      841 gtgcatacag tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat
      901 gagacaaacg cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag
      961 agcgcacgag ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1021 gccaccactg atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga
     1081 aaaacggctt tgccgcggcc ctctcggatc tgtatggtgc actctcagta caatctgctc
     1141 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg cctcgacctg
     1201 cagcaattcc aacgccatca aaaataattc gcgtctggcc ttcctgtagc cagctttcat
     1261 caacattaaa tgtgagcgag taacaacccg tcggattctc cgtgggaaca aacggcggat
     1321 tgaccgtaat gggataggtc acgttggtgt agatgggcgc atcgtaaccg tgcatctgcc
     1381 agtttgaggg gacgacgaca gtatcggcct caggaagatc gcactccagc cagctttccg
     1441 gcaccgcttc tggtgccgga aaccaggcaa agcgccattc gccattcagg ctgcgcaact
     1501 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     1561 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     1621 cgacggccag tgaatccgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1681 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1741 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1801 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1861 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     1921 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     1981 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     2041 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     2101 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     2161 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     2221 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     2281 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     2341 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     2401 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     2461 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     2521 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     2581 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     2641 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     2701 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     2761 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     2821 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     2881 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattccatgg tgtcggaatt
     2941 gctgcaggtc gagggggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3001 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3061 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagg atggcgccca
     3121 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     3181 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     3241 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc cgtcgaccta
     3301 cagggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc
     3361 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
     3421 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc
     3481 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg
     3541 ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat
     3601 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     3661 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     3721 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     3781 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     3841 gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
     3901 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     3961 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     4021 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     4081 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     4141 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     4201 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     4261 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     4321 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     4381 ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     4441 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
     4501 agattttgag acacaacgtg gctttccatc aaaaaaatat tgacaacata aaaaactttg
     4561 tgttatactt gtggaattgt gagcggataa caattctata tctgttattt tttccaacca
     4621 cagatctatg agcaaaggtg aagaactgtt taccggcagc gagctgatta aggagaacat
     4681 gcacatgaag ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga
     4741 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg
     4801 ccctctcccc ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt
     4861 catcaaccac acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac
     4921 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
     4981 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa
     5041 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc
     5101 cgctgacggc ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca
     5161 tctgatcgca aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat
     5221 gcctggcgtc tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac
     5281 ctacgtcgag cagcacgagg tggcagtg
//
