LOCUS       40924_26864        6608 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pKLD66, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6608)
  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 6608)
  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 6608)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6608)
  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..6608
                     /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             1251..1268
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      1335..1382
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1419..1874
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1901..2005
                     /label=AmpR promoter
     CDS             2006..2863
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3037..3625
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(3811..3953)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4058..4246)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5021..5042)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5058..6137)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6138..6215)
                     /label=lacI promoter
     promoter        6524..6542
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6543..6567
                     /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             6582..6604
                     /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 accacgtcag agaatctcta cttccaaggt
     1201 acctcggact ctagagtcga cctgcaggca agcttgcggc cgcactcgag caccaccacc
     1261 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1321 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1381 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1441 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1501 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1561 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1621 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1681 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1741 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1801 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1861 aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1921 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1981 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2041 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2101 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2161 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2221 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2281 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     2341 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     2401 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     2461 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     2521 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     2581 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     2641 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     2701 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2761 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2821 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2881 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2941 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3001 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3061 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3121 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3181 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3241 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3301 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3361 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3421 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3481 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3541 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3601 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3661 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3721 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3781 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3841 gtgcggtatt tcacaccgca tatatggtgc actctcagta caatctgctc tgatgccgca
     3901 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3961 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     4021 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     4081 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     4141 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     4201 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     4261 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     4321 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4381 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4441 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4501 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4561 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4621 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4681 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4741 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4801 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4861 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4921 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4981 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     5041 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5101 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     5161 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     5221 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     5281 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     5341 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5401 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5461 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5521 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5581 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5641 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5701 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5761 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5821 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5881 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5941 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     6001 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     6061 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     6121 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     6181 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     6241 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     6301 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     6361 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     6421 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     6481 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     6541 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     6601 gagatata
//
