LOCUS       pET-28b(+)NeqXX7        7873 bp DNA     circular SYN 25-JAN-2023
DEFINITION  Bacterial vector for expression of N-terminally 6xHis-tagged 
            proteins with a thrombin site. For other reading frames, use 
            pET-28a(+) or pET-28c(+).
ACCESSION   .
VERSION     .
KEYWORDS    pET-28b(+)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7873)
  AUTHORS   MilliporeSigma (Novagen)
  TITLE     Direct Submission
  JOURNAL   Exported Jan 25, 2023 from SnapGene 6.2.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7873
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      26..73
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    158..163
                     /label=MCS
                     /note="multiple cloning site"
     CDS             complement(2783..2800)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2810..2832
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2847..2871
                     /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(2872..2890)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3199..3276
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3277..4359
                     /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"
     CDS             5168..5359
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(5789..6377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             6499..7314
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(7407..7862)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctacttt ttctgcttct
      181 ccagcatctg cagcagctcc ttcggcgcgt ccttttcgct taccgcacca cggccggtct
      241 tgccaccgcc ctcgtcgtag gtgaaggaga tcatcttgcc cacacgccat actttcttga
      301 tcttggagat gtctacctct ttttcttcgc ctttgtactt gaactttacg gttgcaccgc
      361 caccgccggt acctttaaag aaatctgtta gttttttgcc tgtgaatgtg tctttaatac
      421 ctacagattc taatatttgc tctactgcag gcaatagttg tttttctatg tagtattcgg
      481 ggtcatattc attaccccta acttctttgt ctattttaat tctatctgaa acccttccag
      541 tgctacctac cactaaatat ctaactctaa agccaggcat aactttatag cccaaccttt
      601 tatatttttt tgctgctgcc acatgaggag cttcaacttt gtatttttct aaaggctttc
      661 ttatcgtttc tgtaattaaa agcttctccc atggtatttc tctatttctt agctttctaa
      721 taatttcttt tatgtattgt cttgcttttt ctatgttacc ttcttttagc aaaatttcca
      781 atagctttgt ttggacttct ttaactatgg gagcccagtc tgttcttact gcttcgaagc
      841 cccttagctt tatgtaacct tgttcgctta ataaagcata tttctttttt gccccctttt
      901 gttcacccct cttttttacg aatatccctc taacatagaa atcttctaaa tctaattgta
      961 ttttaccggg taattcttta tttactttct ctaaaaattc taatactttg tctttgtccc
     1021 cagaaatgaa taatgaatcg gtatctccat aaattacttt gaatccttct tcttcggccc
     1081 tttttaatat ataattaatg tatttcctgc cccaagctgc tactgccgca gcacagtcta
     1141 tgcaatacca tctcgctctt gggaaaccca tatagccata ggttgcatta attattattt
     1201 tcaatactct ttgcttagca tttataattt tatattcttc actatcccta tctagttctt
     1261 tcagttttct cttaagttct agcctttttg tatacaaatg ctctaatacc gttggaataa
     1321 acccttttac ttttttgcaa aaccacacat attttttctc attatttatt atcgcttcta
     1381 ccccattttg tttacaatca tcatgctcac aatatattgt gcctggagaa acattatgct
     1441 ctaatataat tgaaggatac agagaagcga aatccagtac agctaaattc tcatacaaac
     1501 cgggttttgg ttcgtaaacg aatgctcctg cataggtatt taattttctt ctctctactt
     1561 cttccatttt tggtttgtta ggcacaattt cattatattt tctgcttttt ttgattagat
     1621 accattcaac tatattacta tagctaaacc tagatacatc aaataatggt tggttaacca
     1681 atcttgctaa ttccaataaa tcattttcat agtatttgaa tagcttatat gttaattcgg
     1741 catcttttaa attgtattcg aacactttgt tataattgcc tatggcccaa tcatataaca
     1801 tgttttcata atcataaatt ttctcctctc caagaatttc tttagctact tcatttaatg
     1861 aatatgtttg ggtagacaat tgggttttat atatggtagt aattgtttta tacaaatcta
     1921 catgtgcgct ttccctaaaa ttataagtag gctctttttt gttaaccaat tttattggta
     1981 gccttatatt gtataaagaa gcccttttca ctaaaaagtc tatatcgaaa ttatccccat
     2041 tgtatgtaac tattatgtct ggttttaagc tttctattat ttcgacaaat ttctcaatta
     2101 attcatattc ggtattaact tttatagttg gttctccttt gtaagagata accccttcgt
     2161 aattgtctcc ataaagggct atagatatta ttttgtcttt ttcaggatta gggaaagcct
     2221 cactgtaaac ttctatatca aaagctaaaa ccgatggctt agtcttttca tacaatcttt
     2281 ttatactttg tgcttttatt ctaactttgt tagaggtaga taataaaatt ttgccttcta
     2341 cctcaaacca ttcaaaaggc tctatccctt tatctattaa aaacttccta taaacattta
     2401 tatcggtttc gtatttttta tagccctttt ctttaatgaa ttcttttagt attggataat
     2461 gtttatgcaa aacatatact ttataaacat cttttggttt ttctatatat ttcttttgct
     2521 cctttgtata ttcgattttt atttttttgt tgttagcgaa ttcttctaaa tcctttatat
     2581 cgccttcaaa atagaaatac catttgtatg gaattataac ttcacaagct ctattatttt
     2641 cgtctttacc tattaaaatt acctcgtcta aaggggtgtt ctctatatta atccaataac
     2701 attttagcac gctataccct tcttcctctt ttaccgcctt ttcttctaca accatcgtgg
     2761 ggagttggtg taacattgat ccgtgatggt gatggtgatg catggtatat ctccttctta
     2821 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     2881 agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc atcgtggccg
     2941 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     3001 aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag
     3061 gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg
     3121 cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg
     3181 gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     3241 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     3301 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     3361 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     3421 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     3481 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     3541 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     3601 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     3661 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     3721 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     3781 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     3841 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     3901 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     3961 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     4021 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     4081 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     4141 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     4201 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     4261 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4321 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     4381 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     4441 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     4501 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     4561 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     4621 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     4681 tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     4741 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     4801 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     4861 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     4921 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     4981 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     5041 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     5101 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     5161 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     5221 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     5281 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga
     5341 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     5401 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     5461 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
     5521 tcagagcaga ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg
     5581 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5641 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5701 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5761 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5821 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5881 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5941 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6001 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6061 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6121 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6181 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     6241 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6301 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6361 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6421 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac
     6481 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     6541 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     6601 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     6661 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     6721 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     6781 tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct
     6841 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     6901 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     6961 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     7021 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     7081 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     7141 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     7201 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     7261 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     7321 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     7381 cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc
     7441 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     7501 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     7561 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     7621 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     7681 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     7741 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     7801 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     7861 gtcccattcg cca
//
