LOCUS       pET28a-pT5-T7RNAP       7912 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  Nucleus PURE: pET28a-pT5-T7RNAP in pET28a protein expression
            plasmid. See https://nucleus.bnext.bio/ for more details..
ACCESSION   pET28a-pT5-T7RNAP
VERSION     pET28a-pT5-T7RNAP
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%"
     source          3561..6357
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        3561..3605
                     /label="T5 promoter"
     protein_bind    3581..3597
                     /label="lac operator"
     protein_bind    3613..3629
                     /label="lac operator"
     RBS             3648..3659
                     /label="RBS"
     CDS             3666..3677
                     /codon_start=1
                     /label="N-term"
                     /translation="MRGS"
     CDS             3678..3695
                     /codon_start=1
                     /label="6xHis"
                     /translation="HHHHHH"
     CDS             3696..3701
                     /codon_start=1
                     /label="Linker"
                     /translation="GS"
     CDS             3702..6350
                     /codon_start=1
                     /label="T7RNAP"
                     /translation="MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYE
                     MGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQ
                     FLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFK
                     KNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIE
                     STGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGI
                     TGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANV
                     ITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLE
                     FMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYY
                     WLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFE
                     YAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAK
                     KVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVM
                     TLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVE
                     AMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLG
                     QFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHD
                     SFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLN
                     LRDILESDFAFA"
     terminator      6421..6468
                     /label="T7 terminator"
                     /note="Color: #ffffff NCBI Feature Key: terminator
                     Transferred From: T7_terminator Transferred Similarity:
                     100.00%"
     rep_origin      6505..6960
                     /direction=RIGHT
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(7052..7867)
                     /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 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta
     3601 taatagattc aattgtgagc ggataacaat ttcacacaga attcattaaa gaggagaaat
     3661 taactatgag aggatcgcat caccatcacc atcacggatc catgaacacg attaacatcg
     3721 ctaagaacga cttctctgac atcgaactgg ctgctatccc gttcaacact ctggctgacc
     3781 attacggtga gcgtttagct cgcgaacagt tggcccttga gcatgagtct tacgagatgg
     3841 gtgaagcacg cttccgcaag atgtttgagc gtcaacttaa agctggtgag gttgcggata
     3901 acgctgccgc caagcctctc atcactaccc tactccctaa gatgattgca cgcatcaacg
     3961 actggtttga ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc cagttcctgc
     4021 aagaaatcaa gccggaagcc gtagcgtaca tcaccattaa gaccactctg gcttgcctaa
     4081 ccagtgctga caatacaacc gttcaggctg tagcaagcgc aatcggtcgg gccattgagg
     4141 acgaggctcg cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag aaaaacgttg
     4201 aggaacaact caacaagcgc gtagggcacg tctacaagaa agcatttatg caagttgtcg
     4261 aggctgacat gctctctaag ggtctactcg gtggcgaggc gtggtcttcg tggcataagg
     4321 aagactctat tcatgtagga gtacgctgca tcgagatgct cattgagtca accggaatgg
     4381 ttagcttaca ccgccaaaat gctggcgtag taggtcaaga ctctgagact atcgaactcg
     4441 cacctgaata cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc atctctccga
     4501 tgttccaacc ttgcgtagtt cctcctaagc cgtggactgg cattactggt ggtggctatt
     4561 gggctaacgg tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa gcactgatgc
     4621 gctacgaaga cgtttacatg cctgaggtgt acaaagcgat taacattgcg caaaacaccg
     4681 catggaaaat caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag tggaagcatt
     4741 gtccggtcga ggacatccct gcgattgagc gtgaagaact cccgatgaaa ccggaagaca
     4801 tcgacatgaa tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct gtgtaccgca
     4861 aggacaaggc tcgcaagtct cgccgtatca gccttgagtt catgcttgag caagccaata
     4921 agtttgctaa ccataaggcc atctggttcc cttacaacat ggactggcgc ggtcgtgttt
     4981 acgctgtgtc aatgttcaac ccgcaaggta acgatatgac caaaggactg cttacgctgg
     5041 cgaaaggtaa accaatcggt aaggaaggtt actactggct gaaaatccac ggtgcaaact
     5101 gtgcgggtgt cgataaggtt ccgttccctg agcgcatcaa gttcattgag gaaaaccacg
     5161 agaacatcat ggcttgcgct aagtctccac tggagaacac ttggtgggct gagcaagatt
     5221 ctccgttctg cttccttgcg ttctgctttg agtacgctgg ggtacagcac cacggcctga
     5281 gctataactg ctcccttccg ctggcgtttg acgggtcttg ctctggcatc cagcacttct
     5341 ccgcgatgct ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct agtgaaaccg
     5401 ttcaggacat ctacgggatt gttgctaaga aagtcaacga gattctacaa gcagacgcaa
     5461 tcaatgggac cgataacgaa gtagttaccg tgaccgatga gaacactggt gaaatctctg
     5521 agaaagtcaa gctgggcact aaggcactgg ctggtcaatg gctggcttac ggtgttactc
     5581 gcagtgtgac taagcgttca gtcatgacgc tggcttacgg gtccaaagag ttcggcttcc
     5641 gtcaacaagt gctggaagat accattcagc cagctattga ttccggcaag ggtctgatgt
     5701 tcactcagcc gaatcaggct gctggataca tggctaagct gatttgggaa tctgtgagcg
     5761 tgacggtggt agctgcggtt gaagcaatga actggcttaa gtctgctgct aagctgctgg
     5821 ctgctgaggt caaagataag aagactggag agattcttcg caagcgttgc gctgtgcatt
     5881 gggtaactcc tgatggtttc cctgtgtggc aggaatacaa gaagcctatt cagacgcgct
     5941 tgaacctgat gttcctcggt cagttccgct tacagcctac cattaacacc aacaaagata
     6001 gcgagattga tgcacacaaa caggagtctg gtatcgctcc taactttgta cacagccaag
     6061 acggtagcca ccttcgtaag actgtagtgt gggcacacga gaagtacgga atcgaatctt
     6121 ttgcactgat tcacgactcc ttcggtacca ttccggctga cgctgcgaac ctgttcaaag
     6181 cagtgcgcga aactatggtt gacacatatg agtcttgtga tgtactggct gatttctacg
     6241 accagttcgc tgaccagttg cacgagtctc aattggacaa aatgccagca cttccggcta
     6301 aaggtaactt gaacctccgt gacatcttag agtcggactt cgcgttcgcg taagcttgat
     6361 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     6421 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     6481 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     6541 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     6601 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     6661 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     6721 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     6781 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     6841 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6901 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     6961 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     7021 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     7081 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     7141 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     7201 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     7261 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     7321 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     7381 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     7441 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     7501 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     7561 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     7621 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     7681 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     7741 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     7801 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     7861 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gt
//
