LOCUS       pET28a-RF3              6836 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-RF3 in pET28a protein expression plasmid. See
            https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-RF3
VERSION     pET28a-RF3
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     rep_origin      77..665
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     promoter        77..105
                     /label="RNAII promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter NCBI Join
                     Type: join Transferred From: RNAII_promoter Transferred
                     Similarity: 100.00%"
     misc_RNA        113..665
                     /label="RNAII transcript (complementary strand)"
                     /note="RNAII transcript (complementary strand)"
     misc_RNA        complement(116..223)
                     /label="RNAI transcript"
                     /note="RNAI transcript"
     promoter        complement(230..259)
                     /label="RNA I promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter NCBI Join
                     Type: join Transferred From: RNA_I_promoter Transferred
                     Similarity: 100.00%"
     terminator      669..712
                     /label="rop/bom terminator"
     misc_binding    752..760
                     /label="dnaA binding site"
                     /bound_moiety="dnaA"
     misc_feature    785..848
                     /label="L-strand Y effector site"
                     /note="L-strand Y effector site"
     misc_feature    851..990
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     misc_feature    1031..1187
                     /label="H-strand Y effector site"
                     /note="H-strand Y effector site"
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /label="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     RBS             1284..1293
     misc_feature    complement(1359..1377)
                     /label="ROP calculated promoter"
     CDS             complement(2092..3174)
                     /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"
     promoter        complement(3175..3252)
                     /label="lacI promoter"
                     /note="Color: #ffffff NCBI Feature Key: promoter
                     Transferred From: lacI_promoter Transferred Similarity:
                     100.00%"
     promoter        3561..3579
                     /label="T7_promoter"
     protein_bind    3580..3604
                     /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)."
     RBS             3613..3645
                     /label="UTR1"
                     /note="note: UTR1 NCBI Feature Key: misc_feature Color:
                     #cc0000 modified by: Andrey Transferred From: U4m - UTR1
                     Transferred Similarity: 100.00%"
     CDS             3649..3651
                     /codon_start=1
                     /label="Start"
                     /translation="M"
     CDS             3652..3669
                     /codon_start=1
                     /label="His6"
                     /translation="HHHHHH"
     CDS             3670..3684
                     /codon_start=1
                     /label="Linker"
                     /translation="GGGGS"
     CDS             3685..5271
                     /codon_start=1
                     /standard_name="RF3"
                     /label="RF3"
                     /translation="MTLSPYLQEVAKRRTFAIISHPDAGKTTITEKVLLFGQAIQTAGT
                     VKGRGSNQHAKSDWMEMEKQRGISITTSVMQFPYHDCLVNLLDTPGHEDFSEDTYRTLT
                     AVDCCLMVIDAAKGVEDRTRKLMEVTRLRDTPILTFMNKLDRDIRDPMELLDEVENELK
                     IGCAPITWPIGCGKLFKGVYHLYKDETYLYQSGKGHTIQEVRIVKGLNNPDLDAAVGED
                     LAQQLRDELELVKGASNEFDKELFLAGEITPVFFGTALGNFGVDHMLDGLVEWAPAPMP
                     RQTDTRTVEASEDKFTGFVFKIQANMDPKHRDRVAFMRVVSGKYEKGMKLRQVRTAKDV
                     VISDALTFMAGDRSHVEEAYPGDILGLHNHGTIQIGDTFTQGEMMKFTGIPNFAPELFR
                     RIRLKDPLKQKQLLKGLVQLSEEGAVQVFRPISNNDLIVGAVGVLQFDVVVARLKSEYN
                     VEAVYESVNVATARWVECADAKKFEEFKRKNESQLALDGGDNLAYIATSMVNLRLAQER
                     YPDVQFHQTREH"
     terminator      5345..5392
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      5429..5884
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5976..6791)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1381 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1441 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1501 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1561 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1621 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1681 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1741 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     1801 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     1861 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     1921 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     1981 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     2041 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     2101 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2161 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     2221 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     2281 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     2341 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     2401 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     2461 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     2521 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     2581 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     2641 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     2701 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     2761 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     2821 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     2881 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     2941 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     3001 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     3061 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     3121 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     3181 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     3241 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     3301 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     3361 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     3421 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     3481 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     3541 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     3601 ttcccctcta gaaataattt tgtttaactt taagaaggag atataccaat gcaccatcac
     3661 caccatcatg gaggcggtgg atctatgact ctgtcgccgt atttgcagga agttgcaaaa
     3721 cgtcgtacct tcgcgattat ctcacatcct gatgccggga aaaccactat tacagaaaaa
     3781 gtgctgctgt ttggccaggc gattcaaacc gccggcactg tgaaaggacg tggaagcaac
     3841 cagcatgcta agtcagattg gatggagatg gaaaaacaac gtggtatcag cattacaacc
     3901 agcgtgatgc agttcccgta ccacgactgc ctggtgaacc tgctggatac tccagggcat
     3961 gaggatttct ccgaagatac ataccgcact cttacggcgg tggattgttg cctgatggtc
     4021 atcgatgctg caaaaggagt tgaggatcgg acgcgcaaac tgatggaagt tacccgttta
     4081 cgtgacacgc cgattcttac atttatgaac aagctggatc gtgatattcg ggatccgatg
     4141 gaacttctgg atgaagtcga aaatgaactg aaaatcggtt gtgcaccgat tacgtggccg
     4201 atcggttgtg gtaaactgtt taaaggtgta tatcacctgt ataaggacga aacgtacctg
     4261 taccagagcg gcaaaggcca caccatccaa gaggtgcgta tcgtgaaagg tttgaacaac
     4321 ccggatctgg acgccgctgt tggcgaggat ttagctcagc agctgcgcga cgaactggag
     4381 ctggtgaaag gtgcgtctaa cgaatttgac aaagaattat tcctggcggg tgaaattacc
     4441 ccggtctttt tcgggacggc tctggggaac tttggtgtgg accacatgct ggatgggtta
     4501 gtggaatggg ctccagcccc gatgccgcgg caaacagata cgcggactgt tgaagccagt
     4561 gaggacaaat tcactggatt cgtgtttaag attcaggcga atatggatcc taaacaccgt
     4621 gatcgcgtgg cttttatgcg cgtggtatcc ggcaaatatg agaaggggat gaaattgcgt
     4681 caggtccgga ctgccaagga tgtagttatc agcgacgcct tgacgttcat ggcgggtgat
     4741 cggagtcatg tcgaggaagc gtacccgggg gacattctgg gattacacaa tcatggcact
     4801 attcagatcg gtgacacatt cacccagggc gagatgatga aatttaccgg gattccgaac
     4861 ttcgcacctg aactttttcg ccgtattcgg ctgaaggatc cacttaaaca gaaacagctg
     4921 ctgaaaggtc tggtccaatt gtctgaagaa ggcgcggtgc aggttttccg tccgatctct
     4981 aataatgatc tgatcgtggg tgcggtaggg gttctgcagt tcgacgttgt ggtagcgcgg
     5041 ctgaaatcag agtacaacgt ggaagcagta tacgagtccg ttaatgtcgc tactgcacgc
     5101 tgggtcgaat gcgcggatgc aaaaaaattt gaggaattta aacgcaaaaa cgaaagccaa
     5161 ctggcgttgg acggcggcga taacttagca tatatcgcga ccagtatggt aaatctgcgt
     5221 ctggcccagg aacgctaccc agacgtccaa tttcatcaaa cccgtgagca ttgaagagct
     5281 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     5341 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg
     5401 aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     5461 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5521 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5581 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5641 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5701 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5761 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5821 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5881 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5941 tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa
     6001 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     6061 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     6121 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     6181 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt
     6241 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     6301 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga
     6361 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     6421 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     6481 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     6541 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     6601 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     6661 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     6721 tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga
     6781 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgt
//
