LOCUS       40924_26859        6626 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLD116, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6626)
  AUTHORS   Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena 
            JC.
  TITLE     Construction and use of new cloning vectors for the rapid isolation 
            of recombinant proteins from Escherichia coli
  JOURNAL   Plasmid 59 (3), 231-237 (2008)
  PUBMED    18295882
REFERENCE   2  (bases 1 to 6626)
  AUTHORS   Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena 
            JC.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-DEC-2007) Bacteriology, University of Wisconsin - 
            Madison, 1550 Linden Dr., Madison, WI 53706, USA
REFERENCE   3  (bases 1 to 6626)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6626)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plasmid"; 
            date: "2008"; volume: "59"; issue: "3"; pages: "231-237"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (10-DEC-2007) Bacteriology, University of Wisconsin - Madison, 1550 
            Linden Dr., Madison, WI 53706, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6626
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             13..30
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             37..1134
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     CDS             1180..1200
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQG"
     CDS             1269..1286
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      1353..1400
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1437..1892
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1919..2023
                     /label=AmpR promoter
     CDS             2024..2881
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3055..3643
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(3829..3971)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4076..4264)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5039..5060)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5076..6155)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6156..6233)
                     /label=lacI promoter
     promoter        6542..6560
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6561..6585
                     /label=lac operator
                     /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             6600..6622
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catatgggca gccatcacca tcaccatcac tccggtaaaa tcgaagaagg taaactggta
       61 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
      121 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
      181 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
      241 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
      301 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
      361 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
      421 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
      481 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
      541 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
      601 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
      661 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
      721 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
      781 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
      841 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
      901 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
      961 gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa
     1021 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     1081 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     1141 agctcgagcg gaaccgcctc gggcggtgca accactagtg agaatctcta cttccaaggc
     1201 cttagcaggt gcatgtggac gtcccgggct agccatggcc tgcaggcaag cttgcggccg
     1261 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1321 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1381 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1441 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1501 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1561 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1621 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1681 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1741 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1801 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1861 gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg
     1921 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1981 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2041 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2101 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2161 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2221 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     2281 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2341 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2401 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2461 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2521 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2581 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2641 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2701 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2761 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     2821 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     2881 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     2941 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3001 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3061 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3121 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3181 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3241 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3301 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3361 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3421 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3481 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3541 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3601 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3661 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3721 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3781 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3841 ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac tctcagtaca
     3901 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     3961 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4021 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4081 tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt
     4141 gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa
     4201 gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg
     4261 tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac
     4321 gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt
     4381 gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc
     4441 aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg
     4501 cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg
     4561 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
     4621 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
     4681 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca
     4741 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     4801 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     4861 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     4921 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     4981 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     5041 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5101 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5161 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     5221 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     5281 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     5341 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     5401 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     5461 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     5521 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     5581 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     5641 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     5701 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     5761 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     5821 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     5881 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     5941 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     6001 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     6061 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     6121 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     6181 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     6241 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     6301 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     6361 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     6421 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     6481 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     6541 ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt
     6601 ttgtttaact ttaagaagga gatata
//
