LOCUS       pET28a-RRF              5801 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-RRF in pET28a protein expression plasmid. See
            https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-RRF
VERSION     pET28a-RRF
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..4203
                     /codon_start=1
                     /standard_name="RRF"
                     /label="RRF"
                     /translation="MISDIRKDAEVRMDKCVEAFKTQISKIRTGRASPSLLDGIVVEYY
                     GTPTPLRQLASVTVEDSRTLKINVFDRSMSPAVEKAIMASDLGLNPNSAGSDIRVPLPP
                     LTEERRKDLTKIVRGEAEQARVAVRNVRRDANDKVKALLKDKEISEDDDRRSQDDVQKL
                     TDAAIKKIEAALADKEAELMQF"
     CDS             4204..4218
                     /codon_start=1
                     /label="Linker"
                     /translation="GGGGS"
     CDS             4219..4236
                     /codon_start=1
                     /label="His6"
                     /translation="HHHHHH"
     terminator      4310..4357
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      4394..4849
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(4941..5756)
                     /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 gattagcgat
     3661 atccgtaaag acgcggaagt gcgcatggat aaatgtgttg aggcctttaa gacccagatc
     3721 tctaagatcc ggacgggtcg cgccagcccg agtttactgg atggtatcgt ggtcgaatat
     3781 tatgggacac ctactccgct gcggcaactg gcctcggtta ccgtcgaaga tagccgcacc
     3841 ctgaaaatca acgtattcga tcgctcaatg agccctgccg ttgaaaaagc gatcatggcg
     3901 agcgacctgg gcttaaaccc gaatagtgca gggtctgaca tccgtgtgcc gttgccaccg
     3961 ttgacagaag aacgccgcaa ggaccttact aaaatcgtac gtggtgaagc tgaacaggct
     4021 cgcgtcgccg ttcgtaacgt ccggcgcgac gcgaatgaca aggtgaaagc gttgttgaag
     4081 gacaaagaaa tctccgaaga tgatgatcgt cgcagccagg acgatgtaca gaaactgaca
     4141 gatgcggcga ttaagaaaat tgaagcggcc ctggctgata aagaggctga gctgatgcaa
     4201 tttggaggcg gtggatctca ccatcaccac catcattgaa gagcttgatc cggctgctaa
     4261 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
     4321 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
     4381 attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     4441 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     4501 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     4561 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     4621 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     4681 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     4741 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4801 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     4861 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     4921 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     4981 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     5041 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     5101 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     5161 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     5221 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     5281 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     5341 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     5401 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     5461 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     5521 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     5581 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     5641 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     5701 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca
     5761 ccccttgtat tactgtttat gtaagcagac agttttattg t
//
