LOCUS       pET-28a(+)-8HisG        5369 bp DNA     circular SYN 08-AUG-2024
DEFINITION  Bacterial vector for expression of N-terminally 6xHis-tagged 
            proteins with a thrombin site. For other reading frames, use 
            pET-28b(+) or pET-28c(+).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5369)
  AUTHORS   MilliporeSigma (Novagen)
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2024 from SnapGene Viewer 6.2.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5369
                     /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..203
                     /label=MCS
                     /note="multiple cloning site"
     CDS             complement(207..239)
                     /codon_start=1
                     /product="epitope tag from a T7 major capsid protein"
                     /label=T7 tag
                     /translation="MASMTGGQQMG"
     CDS             complement(243..260)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(270..287)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(297..299)
                     /codon_start=1
                     /product="start codon"
                     /label=ATG
                     /translation="M"
     RBS             307..312
                     /note="ribosome binding site"
     protein_bind    343..367
                     /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(368..386)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        695..772
                     /gene="lacI"
                     /label=lacI promoter
     CDS             773..1855
                     /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             2664..2855
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3285..3873)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             3995..4810
                     /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(4903..5358)
                     /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 gagtgcggcc gcaagcttgt
      181 cgacggagct cgaattcgga tccgcgaccc atttgctgtc caccagtcat gctagccata
      241 tggctgccgc gcggcaccag gccgctgctg tgatgatgat gatgatggtg gtgtttcatg
      301 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
      361 caattcccct atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc
      421 ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc
      481 gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc
      541 gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca
      601 ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg
      661 caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt
      721 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
      781 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
      841 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
      901 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
      961 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     1021 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     1081 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     1141 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     1201 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     1261 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     1321 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     1381 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     1441 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     1501 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     1561 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
     1621 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     1681 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
     1741 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     1801 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     1861 acgcaattaa tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga
     1921 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1981 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     2041 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     2101 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     2161 gcgagaagca ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc
     2221 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     2281 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     2341 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     2401 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     2461 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     2521 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     2581 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     2641 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     2701 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     2761 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc
     2821 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2881 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2941 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     3001 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatat gcggtgtgaa
     3061 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3121 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3181 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3241 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3301 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3361 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3421 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3481 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3541 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3601 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3661 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3721 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3781 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3841 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3901 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact
     3961 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     4021 ttgctctagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     4081 tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc
     4141 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     4201 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     4261 tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt
     4321 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     4381 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     4441 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     4501 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     4561 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     4621 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     4681 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     4741 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     4801 gtttttctaa gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac
     4861 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgaaattgta aacgttaata
     4921 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4981 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     5041 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     5101 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     5161 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5221 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5281 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5341 cgccgctaca gggcgcgtcc cattcgcca
//
