LOCUS       Exported                9154 bp ds-DNA     circular SYN 07-JUN-2012
DEFINITION  attB1-attB2.
ACCESSION   .
VERSION     .
KEYWORDS    Expression Clone/pET-DEST42/pDONR221-Dre4-RA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9154)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Apr 15, 2021 from SnapGene 5.2.4
            https://www.snapgene.com
COMMENT     Storage Type: Constructed
FEATURES             Location/Qualifiers
     source          1..9154
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1
                     /label=Gateway Cloning Description
                     /note="Mix Entry Clone pDONR221-Dre4-RA and Destination 
                     Vector pET-DEST42 in LR buffer and TE buffers	Thaw, vortex 
                     and add LR Clonase enzyme (LR Clonase Plus enzyme for 
                     multi-site Gateway). Incubate >= 1 hr. at 25?.	Add 
                     Proteinase K for 10 min at 37?.	Transform into DH5a E. coli
                     cells (One Shot TOP10 for multi-site Gateway) or any non-F'
                     episome bearing strain.	Select Expression Clones on agar 
                     plates with antibiotic ampicillin.	Express in appropriate 
                     host cells."
     misc_feature    34..75
                     /label=V5 epitope
     misc_feature    85..102
                     /label=6x his
     terminator      117..245
                     /label=T7 terminator
     CDS             645..1505
                     /label=Amp(R)
     CDS             1650..2323
                     /label=pBR322 ori
     CDS             complement(2694..2885)
                     /label=ROP
     promoter        5666..5682
                     /label=T7 Promoter
     operon          5685..5709
                     /label=lacO
     misc_feature    5751..5771
                     /label=attB1
     CDS             5777..9142
                     /label=Dre4-RA
     misc_feature    complement(join(9147..9154,1..13))
                     /label=attB2
     misc_feature    9154
                     /label=Gateway Cloning Description
                     /note="Generate PCR product using computed primers and a 
                     DNA polymerase (i.e. Platinum Pfx or Platinum Taq).	Check 
                     quality and purify product by PEG/Mg2+ precipitation and 
                     agarose gels.	Mix purified PCR product and pDONR Vector 
                     pDONR221 in BP buffer and TE buffers.	Thaw, vortex and add 
                     BP Clonase enzyme. Incubate >= 1 hr. at 25?.	Add Proteinase
                     K for 10 min at 37?.	Transform into DH5a or TOP10 cells and
                     plate on kanamycin plates.	Pick colonies and grow in 
                     selection media."
ORIGIN
        1 ttgtacaaag tggtgatcaa ttcgaagctt gaaggtaagc ctatccctaa ccctctcctc
       61 ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagtttg atccggctgc
      121 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      181 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      241 cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
      301 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
      361 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
      421 caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
      481 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
      541 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
      601 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
      661 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
      721 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
      781 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
      841 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
      901 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
      961 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1021 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     1081 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     1141 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     1201 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     1261 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     1321 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     1381 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     1441 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     1501 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     1561 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     1621 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1681 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1741 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1801 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1861 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1921 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1981 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2041 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2101 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2161 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2221 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2281 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2341 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2401 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2461 attttctcct tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac
     2521 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     2581 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     2641 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     2701 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     2761 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     2821 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     2881 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     2941 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3001 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3061 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3121 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3181 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3241 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3301 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     3361 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     3421 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     3481 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     3541 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     3601 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     3661 gtccagtgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     3721 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     3781 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     3841 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     3901 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     3961 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4021 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4081 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4141 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4201 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4261 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4321 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4381 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4441 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4501 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4561 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4621 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4681 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4741 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4801 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4861 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     4921 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     4981 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5041 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5101 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5161 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5221 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5281 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5341 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5401 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5461 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5521 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5581 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5641 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
     5701 aacaattccc ctctagaaat aattttgttt aactttaaga aggaatatca caagtttgta
     5761 caaaaaagca ggcttaatgt cgagctttgt gctggacaag gaggcgttcg tgcgccgcgt
     5821 gaaacgcctc tacacggaat ggagggctcc cagcatcgga cacgatgacg cgctcaggaa
     5881 tctggactgc atcatgagca tcgtgggcgt cgaagaggat gtcatgtact ccaagtccat
     5941 ggccctgcag ctctggctgc tgggctacga gctgacggac accatctcag tgttttgctc
     6001 ggatgccgtg tattttctca ctagcaaaaa gaaaatagaa ttcctgaagc agacgcagaa
     6061 catcaccgaa gaaggttttc cggaaatcaa tctgcttgtt agggatcgga cggacaagga
     6121 ccagggcaac ttcgagaagc tcatcaaagc cctgcaaaac tcgaagaagg gcaagcgttt
     6181 gggcgtcttc gccaaggacg cctatcccgg ggagttcagc gaagcatgga agaaatccct
     6241 gacggcgtcc aagttcgagc atgtggacat tagcactatt atcgcctacc tcatgtgtcc
     6301 caaggatgag tcggagatca acaacatacg caaggcatcg ctagtgtcaa tggacatttt
     6361 caacaagtat ttgaaagatg agatcatgga cattattgat tcggatagga aagtcaaaca
     6421 caacaagctc tcagatggct gcgaggctgc cattggcgag aagaagtaca ccagtggtct
     6481 ggatcccaga ctcctcgata tggcctaccc acccatcatc caatcgggag gagcctacag
     6541 cctgaagttc tccgcggtgg cggacaaaaa tcccttgcac ttcggtgtga ttgtgtgctc
     6601 cttgggcgcc cgctacaagt cgtattgctc aaatatatcg cgcacctttc tggtgaatcc
     6661 caccgaggcc atgcaggaga actacacatt tctggtaagt gtgcaggagg agattctcaa
     6721 gctgctggtt ccgggcacaa agctgtgcga cgtctacgaa aagaccctgg acttcgtgaa
     6781 gaaggagaag ccgagtatgg tggacaatct gcccaaaagc tttggtttcg ccatgggcct
     6841 agagttccgg gagaactcca ttgtcatcgg acccaaatgc caggcgctac tcaagaagaa
     6901 tatggtcttt aatttgcacg tgggcatctc gaatctgacc aatcccgaag ccaccgacaa
     6961 agagggcaag aactacgccc tcttcatcgg agacactgtg ctagtaggcg aacaatcccc
     7021 agccagtgtg atgactccgt ccaagaagaa gatcaagaac gtcggcatct ttatcaagga
     7081 cgacagcgac gaggaagatg tggacgacaa gaagaccgct aaagaagatc agggcaccga
     7141 gatcctgggc cgcagcaagc gcaacgcggt gctggagtca aagctgcgaa acgagatcaa
     7201 cacggaggag aagcgcaagg agcaccagcg cgagctggct cagcagctga acgagcgcgc
     7261 caaggatcgg ctggccaggc agggaaactc gaaggaggtc gagaaagtgc gcaagaacac
     7321 cgtctcctac aagtccatca gccaaatgcc gcgcgagccg gaagtcaagg agctcaagct
     7381 ttacgtggat aagaagtacg agaccgtcat catgccagtg ttcggcatcc aggtgccgtt
     7441 ccatatatcc accatcaaga acatctcgca gtcggtggag ggcgagtaca cgtacttgcg
     7501 tatcaacttc tttcatcccg gcgccacaat gggtcgcaac gaaggcggac tctatcctca
     7561 gccagaggcc acatttgtca aggaagttac ataccgcagc tctaatgtta aggaacacgg
     7621 ggaggtggga gctccatccg caaatctaaa caatgctttc cggctgatca aggaggtgca
     7681 gaagcgcttc aagacccgcg aggctgagga gcgcgaaaag gaggatcttg tgaagcaaga
     7741 cacactcatt ctgtcccaga acaagggtaa tcccaagctg aaggacctat acattcggcc
     7801 caacatcgtg accaagcgca tgacgggcag cctggaagcg cacagcaatg gattccgcta
     7861 catctccgtg cgcggcgaca aggtggacat cttgtacaac aacatcaaaa gcgcgttttt
     7921 ccagccctgc gatggcgaga tgatcattct tctgcatttc cacctgaagt acgccattat
     7981 gtttggcaaa aagaagcacg tggatgtaca gttctacacg gaagtgggag agatcaccac
     8041 ggatctgggc aagcaccagc acatgcacga ccgcgacgac ttggctgctg agcaggcaga
     8101 acgtgagctg cggcacaagc tcaagactgc cttcaagagc ttctgcgaga aggtggaaac
     8161 catgaccaag tcggtagtgg agttcgacac gccgttccgt gagttgggat tccctggagc
     8221 acccttccgc agcacggtga cgctgcaacc cacctcgggc tccctggtca atctgacgga
     8281 atggccacca tttgtcatca cgctggacga tgtggagttg gtgcacttcg agcgcgttca
     8341 gttccacttg cgcaacttcg atatgatatt tgtgttcaag gagtacaata agaaggttgc
     8401 catggtgaac gccattccta tgaacatgct ggatcacgtg aaggagtggc tcaactcctg
     8461 cgacatccgc tattcggagg gtgttcaatc cctgaactgg caaaagatca tgaagaccat
     8521 caccgacgat cctgagggat tctttgaaca aggcggctgg acattcttgg acccggaatc
     8581 gggcagcgaa ggcgaaaacg aaaccgccga atccgaagag gacgaggcct acaatcccac
     8641 cgacgccgaa tcggatgagg agtcggacga agattccgag tactcagagg catcagaaga
     8701 cagcgaggaa agcgatgagg accttggctc tgatgaggaa tctggcaagg attggtccga
     8761 tctggagcgc gaggctgctg aggaagaccg caaccacgat tatgccgcag atgacaaacc
     8821 acgcaacggc aaattcgatt cgaagaagca cggcaagagt tcaaagcaca gtcgccgcga
     8881 ccgcgaggag gctcgatcgt cgtcgcacag taaaaagcac aagtcgaact cctcctcgtc
     8941 ctcttcgcat ttaaagtcct ccagctccaa acatggcagc tcatctagcc cgtcgaagtc
     9001 gtcgaaggac aagtacaaca gccgggacaa gcatcattcc tcctcatcgt ccggaaacaa
     9061 gagcagcagc aaggacaagg atcgcaagcg ctcgcgggat gacagtcgcg acaatgggca
     9121 caagtcgaag aagtcgcgac attacccagc tttc
//
