LOCUS       40924_16999        7528 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Gateway(R) destination vector for constitutive high-level expression
            of proteins in mammalian cells using the Flp-In(TM) system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7528)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 7528)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        353..1531
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     promoter        1548..1566
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1645..1769
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1794..1824
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1878..2534
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2879..3181
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3225..3349)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3402..3443
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    3487..3711
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3994..4041
                     /label=FRT
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4049..5068
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5201..5334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5371..5387)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5395..5411)
                     /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(5419..5449)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5464..5485)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5773..6361)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6535..7392)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7393..7497)
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac taggcttttg caaaaagctt tgcaaagatg gataaagttt taaacagaga
      301 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgcctcgt gaggctccgg
      361 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      421 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      481 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      541 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      601 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      661 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      721 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      781 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      841 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
      901 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
      961 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1021 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1081 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1141 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1201 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1261 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1321 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1381 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1441 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1501 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtactaa tacgactcac
     1561 tatagggaga cccaagctgg ctaggtaagc ttggtaccga gctcggatcc actagtccag
     1621 tgtggtggaa ttctgcagat atcaacaagt ttgtacaaaa aagctgaacg agaaacgtaa
     1681 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     1741 tgtaaaacac aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac
     1801 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatccggcg agattttcag
     1861 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     1921 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     1981 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     2041 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
     2101 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     2161 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     2221 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     2281 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     2341 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     2401 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     2461 tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     2521 ggcagggcgg ggcgtaaaga tctggatccg gcttactaaa agccagataa cagtatgcgt
     2581 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     2641 caaaaagagg tgtgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     2701 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     2761 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     2821 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacaggga ctggtgaaat
     2881 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     2941 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     3001 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     3061 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     3121 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     3181 ataaatgtca ggctccgtta tacacagcca gtctgcaggt cgaccatagt gactggatat
     3241 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     3301 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtt gatatccagc
     3361 acagtggcgg ccgctcgagt ctagagggcc cgcggttcga aggtaagcct atccctaacc
     3421 ctctcctcgg tctcgattct acgcgtaccg gttagtaatg agtttaaacc cgctgatcag
     3481 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3541 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3601 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     3661 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     3721 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     3781 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3841 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3901 ctctaaatcg ggggtccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3961 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     4021 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     4081 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     4141 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     4201 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     4261 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     4321 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     4381 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     4441 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     4501 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     4561 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     4621 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     4681 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     4741 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     4801 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     4861 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     4921 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     4981 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     5041 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     5101 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5161 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5221 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5281 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5341 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5401 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5461 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     5521 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5581 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5641 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5701 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5761 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5821 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5881 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5941 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6001 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6061 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6121 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6181 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     6241 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6301 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6361 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6421 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6481 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6541 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6601 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6661 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6721 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6781 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6841 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6901 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6961 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7021 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7081 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7141 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7201 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7261 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7321 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7381 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7441 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7501 atttccccga aaagtgccac ctgacgtc
//
