LOCUS       40924_18336        7480 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) destination vector for high-level bacterial expression of
            proteins with a C-terminal tetracysteine (Lumio(TM)) tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7480)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7480)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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             79..101
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             109..141
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     protein_bind    154..278
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        303..333
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             387..1043
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             1388..1690
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(1734..1858)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1887..1904
                     /codon_start=1
                     /label=tetracysteine tag
                     /note="tetracysteine peptide that binds biarsenical
                     labeling reagents"
                     /translation="CCPGCC"
     CDS             1920..1937
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2008..2055
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(2230..2258)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     promoter        2375..2479
                     /label=AmpR promoter
     CDS             2480..3337
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3511..4099
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4285..4427)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4532..4720)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5998..6019)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6035..7114)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7115..7192)
                     /label=lacI promoter
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat ggctagcatg
      121 actggtggac agcaaatggg tattatgatt atcacaagtt tgtacaaaaa agctgaacga
      181 gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact
      241 acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag
      301 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatcctgcga
      361 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
      421 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
      481 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
      541 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
      601 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
      661 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
      721 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
      781 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
      841 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
      901 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
      961 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     1021 gcgatgagtg gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac
     1081 agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc
     1141 gaagtatgtc aaaaagaggt gtgctatgaa gcagcgtatt acagtgacag ttgacagcga
     1201 cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc
     1261 atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg
     1321 atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacagggac
     1381 tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt
     1441 ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag
     1501 tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga
     1561 tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga
     1621 agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt
     1681 ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg
     1741 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     1801 atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtga
     1861 tcaattcgaa gcttgaagct ggtggctgtt gtcctggctg ttgcggtggc ggcaccggtc
     1921 atcatcacca tcaccattga gtttgatccg gctgctaaca aagcccgaaa ggaagctgag
     1981 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     2041 ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca
     2101 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
     2161 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
     2221 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaatta attcttgaag
     2281 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     2341 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     2401 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     2461 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     2521 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     2581 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     2641 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     2701 atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca
     2761 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     2821 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     2881 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     2941 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3001 cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3061 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3121 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     3181 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     3241 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     3301 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     3361 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     3421 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3481 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3541 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3601 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     3661 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3721 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     3781 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     3841 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     3901 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3961 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4021 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4081 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4141 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4201 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4261 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4321 tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4381 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     4441 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4501 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4561 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     4621 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     4681 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     4741 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     4801 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     4861 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     4921 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     4981 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     5041 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     5101 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     5161 acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg
     5221 tgcggctgct ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat
     5281 tcacagttct ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga
     5341 ggtgccgccg gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc
     5401 ggggaggcag acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct
     5461 cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca gtgatcgaag ttaggctggt
     5521 aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag
     5581 catggcctgc aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa
     5641 ggccatccag cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat
     5701 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     5761 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     5821 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5881 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5941 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     6001 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6061 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     6121 gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg
     6181 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat
     6241 cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc
     6301 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc
     6361 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca
     6421 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg
     6481 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga
     6541 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct
     6601 ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt
     6661 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat
     6721 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt
     6781 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg
     6841 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg
     6901 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca
     6961 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg
     7021 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat
     7081 actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt
     7141 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct
     7201 cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg
     7261 ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg
     7321 gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga
     7381 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
     7441 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
//
