LOCUS       sequence-317805-        5326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS Venus reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5326)
  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 5326)
  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: pNK141f2
FEATURES             Location/Qualifiers
     source          1..5326
                     /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)."
     CDS             89..799
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     ICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             89..799
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     ICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(116..144)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     739..759
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(872..889)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1042..1128
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1220..1247
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      1291..1836
                     /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     1737..1756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1883..1902)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(1908..2405)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     2354..2376
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2368..2385
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2369..2385
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2401..2417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2401..2417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2414..2436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2425..2441
                     /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(2449..2455,2456..2473,2474..2479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             complement(2528..3610)
                     /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     3572..3591
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3611..3688)
                     /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     3808..3830
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3963..3982
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4354..5169)
                     /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(4354..5169)
                     /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     5077..5096
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5161..5180
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggaattgtga gcggataaca attctatatc tgttattttt tccaaccaca gatctatgag
       61 caaaggtgaa gaactgttta ccggcgtcag taaaggagaa gaacttttca ctggagttgt
      121 cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tcagtggaga
      181 gggtgaaggt gatgcaacat acggaaaact tacccttaaa ctgatttgca ctactggaaa
      241 actacctgtt ccatggccaa cacttgtcac tactctgggc tatggtctgc aatgctttgc
      301 cagataccca gatcatatga aacagcatga ctttttcaag agtgccatgc ccgaaggtta
      361 tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac gtgctgaagt
      421 caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg attttaaaga
      481 agatggaaac attcttggac acaaattgga atacaactat aactcacaca atgtatacat
      541 caccgcagac aaacaaaaga atggaatcaa agccaacttc aaaattagac acaacattga
      601 agatggaggc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc
      661 tgtcctttta ccagacaacc attacctgtc ctaccaatct gccctttcga aagatcccaa
      721 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
      781 catggatgaa ctatacaaat aagtcgaccg gctgctaaca aagcccgcgg ccgctgaaga
      841 tcgatctcga cgagtgagag aagattttca gcctgataca gattaaatca gaacgcagaa
      901 gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca
      961 tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtca ccccatgcga
     1021 gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     1081 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     1141 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     1201 gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacag
     1261 agcgtcagac cccttaataa gatgatcttc ttgagatcgt tttggtctgc gcgtaatctc
     1321 ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc tctgagctac
     1381 caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac ttgtcctttc
     1441 agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa ttaccagtgg
     1501 ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga tagttaccgg
     1561 ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc ttggagcgaa
     1621 ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca taacagcgga
     1681 atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc gccaggggga
     1741 aacgcctggt atctttatag tcctgtcggg tttcgccacc actgatttga gcgtcagatt
     1801 tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg gctttgccgc ggccctctcg
     1861 gatctgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     1921 cactccgcta tcgctacgtg actgcctcga cctgcagcaa ttccaacgcc atcaaaaata
     1981 attcgcgtct ggccttcctg tagccagctt tcatcaacat taaatgtgag cgagtaacaa
     2041 cccgtcggat tctccgtggg aacaaacggc ggattgaccg taatgggata ggtcacgttg
     2101 gtgtagatgg gcgcatcgta accgtgcatc tgccagtttg aggggacgac gacagtatcg
     2161 gcctcaggaa gatcgcactc cagccagctt tccggcaccg cttctggtgc cggaaaccag
     2221 gcaaagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     2281 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     2341 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatc cgtaatcatg
     2401 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     2461 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactta cattaattgc
     2521 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2581 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     2641 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     2701 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     2761 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     2821 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     2881 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     2941 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3001 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3061 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     3121 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     3181 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     3241 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     3301 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     3361 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     3421 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3481 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     3541 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     3601 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     3661 gaaaggtttt gcaccattcc atggtgtcgg aattgctgca ggtcgagggg gtcatggctg
     3721 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3781 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3841 catcaccgaa acgcgcgagg caggatggcg cccaacagtc ccccggccac ggggcctgcc
     3901 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     3961 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     4021 acgatgcgtc cggcgtagag gatccgtcga cctacagggg gggggggcgc tgaggtctgc
     4081 ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca tccagccaga
     4141 aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg gtgattttga
     4201 acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca
     4261 actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca gcgtaatgct
     4321 ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
     4381 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     4441 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     4501 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     4561 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     4621 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     4681 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     4741 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     4801 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     4861 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     4921 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     4981 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     5041 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     5101 taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat
     5161 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     5221 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttc
     5281 catcaaaaaa atattgacaa cataaaaaac tttgtgttat acttgt
//
