LOCUS       sequence-317806-        5320 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses orthogonal RBS mPapaya reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5320)
  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 5320)
  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: pNK141g2
FEATURES             Location/Qualifiers
     source          1..5320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(40..1122)
                     /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     1084..1103
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1123..1200)
                     /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     1320..1342
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1475..1494
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1866..2681)
                     /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(1866..2681)
                     /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     2589..2608
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2673..2692
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    2839..2862
                     /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             2924..3634
                     /codon_start=1
                     /product="monomeric yellow fluorescent protein derived from
                     the Zoanthus fluorescent protein zFP538"
                     /label=mPapaya1
                     /note="mammalian codon-optimized"
                     /translation="VSKGEGQSKHGLKEEMTVKYHMEGCVNGHKFVITGEGIGNPFKGK
                     QTANLCVIEGGPLPFSEDILSPGFKYGDRIFTEYPQDIVDYFKNSCPAGYTWERSFLFE
                     DGAVCRCNVDITVSEKENCIYHKSIFRGVNFPADGPVMKKMTTNWEASTEKIVPVPKQG
                     ILKGKVKMYLLLKDGGRYHCQFDTVYKAKSVPSKMPEWHFIQHKLLREDRSDAKNQKWQ
                     LTEHAIAGMDELYK"
     primer_bind     complement(3704..3721)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3874..3960
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4052..4079
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4123..4668
                     /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     4569..4588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4715..4734)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(4740..5237)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGIAAGRGSHVAIAECI
                     LA"
     primer_bind     5186..5208
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5200..5217
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5201..5217
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5233..5249)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5233..5249)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5246..5268)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5257..5273
                     /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(5281..5287,5288..5305,5306..5311))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
       61 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      121 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
      181 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      241 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
      301 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      361 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      421 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      481 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      541 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      601 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
      661 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      721 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      781 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
      841 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
      901 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
      961 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     1021 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     1081 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     1141 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt ccatggtgtc
     1201 ggaattgctg caggtcgagg gggtcatggc tgcgccccga cacccgccaa cacccgctga
     1261 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     1321 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcaggatgg
     1381 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     1441 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     1501 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatccgtc
     1561 gacctacagg gggggggggc gctgaggtct gcctcgtgaa gaaggtgttg ctgactcata
     1621 ccaggcctga atcgccccat catccagcca gaaagtgagg gagccacggt tgatgagagc
     1681 tttgttgtag gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc
     1741 gttgtcggga agatgcgtga tctgatcctt caactcagca aaagttcgat ttattcaaca
     1801 aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat
     1861 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1921 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1981 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     2041 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     2101 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     2161 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     2221 gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     2281 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     2341 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     2401 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2461 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2521 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2581 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2641 cgcggcctcg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     2701 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     2761 catcagagat tttgagacac aacgtggctt tccatcaaaa aaatattgac aacataaaaa
     2821 actttgtgtt atacttgtgg aattgtgagc ggataacaat tctatatctg ttattttttc
     2881 caaccacaga tctatgagca aaggtgaaga actgtttacc ggcgtgagca agggcgaggg
     2941 gcaatcgaag catgggctaa aggaggagat gacggtgaag taccacatgg aaggttgcgt
     3001 taatggtcac aaatttgtca ttactggaga gggcattgga aaccctttta agggtaagca
     3061 aactgcaaat ttgtgtgtga tagaaggagg cccgctgccg ttctcggagg acattttaag
     3121 tccgggtttt aaatatggtg accggatttt cacagagtac ccacaggata ttgtagatta
     3181 cttcaagaac tcatgtccag cgggctatac gtgggaaagg agcttcctct ttgaggacgg
     3241 agcggtctgt cgatgcaacg tggacataac agtctctgaa aaggagaact gcatctatca
     3301 caaaagtatc ttcagagggg tgaattttcc cgccgacggc cccgtaatga agaaaatgac
     3361 cactaattgg gaagctagta ccgagaaaat tgtgcctgtt ccaaagcaag ggatattaaa
     3421 aggaaaggtc aaaatgtacc tattgctgaa ggatggcggt cgttatcatt gccagtttga
     3481 tacggtatat aaagctaaat cagtgccctc caagatgcca gaatggcatt ttatacagca
     3541 taagctcctt agggaggacc gctctgatgc taaaaaccag aaatggcaat taacagaaca
     3601 tgcaatcgca ggcatggacg agctgtacaa gtaagtcgac cggctgctaa caaagcccgc
     3661 ggccgctgaa gatcgatctc gacgagtgag agaagatttt cagcctgata cagattaaat
     3721 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     3781 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     3841 caccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     3901 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3961 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     4021 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     4081 gcgtttctac agagcgtcag accccttaat aagatgatct tcttgagatc gttttggtct
     4141 gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt
     4201 tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc agtcaccaaa
     4261 acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact cctctaaatc
     4321 aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg gactcaagac
     4381 gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc atacagtcca
     4441 gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga caaacgcggc
     4501 cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg cacgagggag
     4561 ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca ccactgattt
     4621 gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa cggctttgcc
     4681 gcggccctct cggatctgta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4741 taagccagta tacactccgc tatcgctacg tgactgcctc gacctgcagc aattccaacg
     4801 ccatcaaaaa taattcgcgt ctggccttcc tgtagccagc tttcatcaac attaaatgtg
     4861 agcgagtaac aacccgtcgg attctccgtg ggaacaaacg gcggattgac cgtaatggga
     4921 taggtcacgt tggtgtagat gggcgcatcg taaccgtgca tctgccagtt tgaggggacg
     4981 acgacagtat cggcctcagg aagatcgcac tccagccagc tttccggcac cgcttctggt
     5041 gccggaaacc aggcaaagcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     5101 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     5161 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     5221 tccgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5281 caacatacga gccggaagca taaagtgtaa agcctggggt
//
