LOCUS       40924_16850        7464 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) destination vector for high-level constitutive expression
            of C-terminally V5- and 6xHis-tagged proteins in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7464)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7464)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        475..1653
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     promoter        1670..1688
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1720..1844
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1869..1899
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1953..2609
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2954..3256
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3300..3424)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3450..3491
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     CDS             3501..3518
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    3547..3771
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3817..4245
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4259..4589
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        4637..4684
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     CDS             4703..5098
                     /codon_start=1
                     /label=BSD
                     /note="blasticidin S deaminase"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5259..5392
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5429..5445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5453..5469)
                     /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)."
     promoter        complement(5477..5507)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5522..5543)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5831..6419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6593..7450)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(join(7451..7464,1..91))
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
      421 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc
      481 ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg
      541 gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
      601 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc
      661 gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt
      721 ggttcccgcg ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc
      781 acctggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
      841 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
      901 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
      961 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     1021 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     1081 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     1141 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     1201 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     1261 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     1321 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     1381 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     1441 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     1501 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     1561 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     1621 caaagttttt ttcttccatt tcaggtgtcg tgaggaatta gcttggtact aatacgactc
     1681 actataggga gacccaagct ggctaggtaa gcttgatcaa caagtttgta caaaaaagct
     1741 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     1801 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca
     1861 ccccaggctt tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc
     1921 cggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca
     1981 ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc
     2041 aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga
     2101 aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc
     2161 atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc
     2221 cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc
     2281 acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa
     2341 acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct
     2401 gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg
     2461 ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc
     2521 aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat gaattacaac
     2581 agtactgcga tgagtggcag ggcggggcgt aaagatctgg atccggctta ctaaaagcca
     2641 gataacagta tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt
     2701 atacccgaag tatgtcaaaa agaggtgtgc tatgaagcag cgtattacag tgacagttga
     2761 cagcgacagc tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc
     2821 acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag
     2881 gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac
     2941 agggactggt gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct
     3001 gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct
     3061 ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat
     3121 cggggatgaa agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat
     3181 cggggaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct
     3241 gatgttctgg ggaatataaa tgtcaggctc cgttatacac agccagtctg caggtcgacc
     3301 atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct
     3361 aatttaatat attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag
     3421 tggttgatct agagggcccg cggttcgaag gtaagcctat ccctaaccct ctcctcggtc
     3481 tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc cgctgatcag
     3541 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3601 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3661 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3721 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3781 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3841 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3901 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3961 ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4021 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4081 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4141 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct
     4201 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
     4261 gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca
     4321 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4381 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     4441 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     4501 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     4561 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     4621 gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca tagtataata
     4681 cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat ccaccctcat
     4741 tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag
     4801 cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg
     4861 gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct
     4921 gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg
     4981 tcgacaggtg cttctcgatc tgcatcctgg gatcaaagcg atagtgaagg acagtgatgg
     5041 acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcta
     5101 agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg
     5161 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5221 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5281 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5341 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5401 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5461 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5521 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5581 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5641 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5701 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5761 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5821 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5881 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5941 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6001 cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg
     6061 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6241 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     6301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6721 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7441 gaatactcat actcttcctt tttc
//
