LOCUS       62056_11205        9638 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9638)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9638
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        771..1476
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1522..1542
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1558..1899
                     /codon_start=1
                     /label=GAL4 activation domain
                     /note="activation domain of the GAL4 transcriptional
                     activator"
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     promoter        1905..1923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1942..1968
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     protein_bind    1975..2099
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2124..2154
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2208..2864
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3209..3511
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3555..3679)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4066..4253
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     CDS             complement(4373..5464)
                     /codon_start=1
                     /label=LEU2
                     /note="3-isopropylmalate dehydrogenase, required for
                     leucine biosynthesis"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(5465..5870)
                     /label=LEU2 promoter
     primer_bind     complement(5912..5928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5936..5952)
                     /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(5960..5990)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6005..6026)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     protein_bind    6081..6114
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(6465..7053)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7227..8084)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8085..8189)
                     /label=AmpR promoter
     rep_origin      8471..9635
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tgcatgcctg caggtcgaga tccgggatcg aagaaatgat ggtaaatgaa ataggaaatc
       61 aaggagcatg aaggcaaaag acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt
      121 tgatatgatg tatttggctt tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca
      181 tgctgactct gtggcggacc cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc
      241 tgtgcccggc ggagtttttt gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg
      301 agattggaga agcaataaga atgccggttg gggttgcgat gatgacgacc acgacaactg
      361 gtgtcattat ttaagttgcc gaaagaacct gagtgcattt gcaacatgag tatactagaa
      421 gaatgagcca agacttgcga gacgcgagtt tgccggtggt gcgaacaata gagcgaccat
      481 gaccttgaag gtgagacgcg cataaccgct agagtacttt gaagaggaaa cagcaatagg
      541 gttgctacca gtataaatag acaggtacat acaacactgg aaatggttgt ctgtttgagt
      601 acgctttcaa ttcatttggg tgtgcacttt attatgttac aatatggaag ggaactttac
      661 acttctccta tgcacatata ttaattaaag tccaatgcta gtagagaagg ggggtaacac
      721 ccctccgcgc tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt
      781 tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga
      841 ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat
      901 agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc
      961 attgatggtg gtacataacg aactaatact gtagccctag acttgatagc catcatcata
     1021 tcgaagtttc actacccttt ttccatttgc catctattga agtaataata ggcgcatgca
     1081 acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat
     1141 gacaaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac agcaccaaca
     1201 gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact ttttccttcc
     1261 ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt ccagttactt
     1321 gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga cctgcaatta
     1381 ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt ctttttctgc
     1441 acaatatttc aagctatacc aagcatacaa tcaactccaa gctttgcaaa gatggataaa
     1501 gcggaattaa ttcccgagcc tccaaaaaag aagagaaagg tcgaattggg taccgccgcc
     1561 aattttaatc aaagtgggaa tattgctgat agctcattgt ccttcacttt cactaacagt
     1621 agcaacggtc cgaacctcat aacaactcaa acaaattctc aagcgctttc acaaccaatt
     1681 gcctcctcta acgttcatga taacttcatg aataatgaaa tcacggctag taaaattgat
     1741 gatggtaata attcaaaacc actgtcacct ggttggacgg accaaactgc gtataacgcg
     1801 tttggaatca ctacagggat gtttaatacc actacaatgg atgatgtata taactatcta
     1861 ttcgatgatg aagatacccc accaaaccca aaaaaagaga tctttaatac gactcactat
     1921 agggcgagcg ccgccatgga gtacccatac gacgtaccag attacgctca tatgacaagt
     1981 ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     2041 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg
     2101 cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga
     2161 ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
     2221 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
     2281 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
     2341 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
     2401 gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat
     2461 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
     2521 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
     2581 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
     2641 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
     2701 acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga
     2761 tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc
     2821 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaacg cgtggatccg
     2881 gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa
     2941 tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat
     3001 tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc
     3061 tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg
     3121 aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct
     3181 tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga
     3241 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
     3301 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta
     3361 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     3421 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     3481 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca
     3541 gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt
     3601 ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt tctcgttcag
     3661 ctttcttgta caaagtggtc tcgagctgca gatgaatcgt agatactgaa aaaccccgca
     3721 agttcacttc aactgtgcat cgtgcaccat ctcaatttct ttcatttata catcgttttg
     3781 ccttctttta tgtaactata ctcctctaag tttcaatctt ggccatgtaa cctctgatct
     3841 atagaatttt ttaaatgact agaattaatg cccatctttt ttttggacct aaattcttca
     3901 tgaaaatata ttacgagggc ttattcagaa gctttggact tcttcgccag aggtttggtc
     3961 aagtctccaa tcaaggttgt cggcttgtct accttgccag aaatttacga aaagatggaa
     4021 aagggtcaaa tcgttggtag atacgttgtt gacacttcta aataagcgaa tttcttatga
     4081 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     4141 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     4201 ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggccggtcg
     4261 aaattcccct accctatgaa catattccat tttgtaattt cgtgtcgttt ctattatgaa
     4321 tttcatttat aaagtttatg tacaaatatc ataaaaaaag agaatctttt taagcaagga
     4381 ttttcttaac ttcttcggcg acagcatcac cgacttcggt ggtactgttg gaaccaccta
     4441 aatcaccagt tctgatacct gcatccaaaa cctttttaac tgcatcttca atggccttac
     4501 cttcttcagg caagttcaat gacaatttca acatcattgc agcagacaag atagtggcga
     4561 tagggttgac cttattcttt ggcaaatctg gagcagaacc gtggcatggt tcgtacaaac
     4621 caaatgcggt gttcttgtct ggcaaagagg ccaaggacgc agatggcaac aaacccaagg
     4681 aacctgggat aacggaggct tcatcggaga tgatatcacc aaacatgttg ctggtgatta
     4741 taataccatt taggtgggtt gggttcttaa ctaggatcat ggcggcagaa tcaatcaatt
     4801 gatgttgaac cttcaatgta ggaaattcgt tcttgatggt ttcctccaca gtttttctcc
     4861 ataatcttga agaggccaaa acattagctt tatccaagga ccaaataggc aatggtggct
     4921 catgttgtag ggccatgaaa gcggccattc ttgtgattct ttgcacttct ggaacggtgt
     4981 attgttcact atcccaagcg acaccatcac catcgtcttc ctttctctta ccaaagtaaa
     5041 tacctcccac taattctctg acaacaacga agtcagtacc tttagcaaat tgtggcttga
     5101 ttggagataa gtctaaaaga gagtcggatg caaagttaca tggtcttaag ttggcgtaca
     5161 attgaagttc tttacggatt tttagtaaac cttgttcagg tctaacacta cctgtacccc
     5221 atttaggacc acccacagca cctaacaaaa cggcatcaac cttcttggag gcttccagcg
     5281 cctcatctgg aagtgggaca cctgtagcgt cgatagcagc accaccaatt aaatgatttt
     5341 cgaaatcgaa cttgacattg gaacgaacat cagaaatagc tttaagaacc ttaatggctt
     5401 cggctgtgat ttcttgacca acgtggtcac ctggcaaaac gacgatcttc ttaggggcag
     5461 acattagaat ggtatatcct tgaaatatat atatatattg ctgaaatgta aaaggtaaga
     5521 aaagttagaa agtaagacga ttgctaacca cctattggaa aaaacaatag gtccttaaat
     5581 aatattgtca acttcaagta ttgtgatgca agcatttagt catgaacgct tctctattct
     5641 atatgaaaag ccggttccgg cgctctcacc tttccttttt ctcccaattt ttcagttgaa
     5701 aaaggtatat gcgtcaggcg acctctgaaa ttaacaaaaa atttccagtc atcgaatttg
     5761 attctgtgcg atagcgcccc tgtgtgttct cgttatgttg aggaaaaaaa taatggttgc
     5821 taagagattc gaactcttgc atcttacgat acctgagtat tcccacagtt ggggatctcg
     5881 actctagcta gaggatcaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5941 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     6001 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     6061 gaaacctgtc gtgccagctg ataacttcgt ataatgtatg ctatacgaag ttattaggtc
     6121 tgaagaggag tttacgtcca gccaagctag cttggctgca ggtcgagcgg ccgcgatccg
     6181 gaacccttaa tataacttcg tataatgtat gctatacgaa gttatcagct gcattaatga
     6241 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     6301 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     6361 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     6421 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     6481 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6541 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6601 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6661 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6721 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6781 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6841 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6901 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6961 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     7021 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     7081 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     7141 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     7201 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     7261 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     7321 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     7381 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     7441 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     7501 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     7561 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     7621 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7681 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7741 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7801 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7861 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7921 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7981 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     8041 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     8101 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     8161 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     8221 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     8281 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     8341 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     8401 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     8461 gtgcaccata acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
     8521 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
     8581 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
     8641 cctattctct agctagaaag tataggaact tcagagcgct tttgaaaacc aaaagcgctc
     8701 tgaagacgca ctttcaaaaa accaaaaacg caccggactg taacgagcta ctaaaatatt
     8761 gcgaataccg cttccacaaa cattgctcaa aagtatctct ttgctatata tctctgtgct
     8821 atatccctat ataacctacc catccacctt tcgctccttg aacttgcatc taaactcgac
     8881 ctctacattt tttatgttta tctctagtat tactctttag acaaaaaaat tgtagtaaga
     8941 actattcata gagtgaatcg aaaacaatac gaaaatgtaa acatttccta tacgtagtat
     9001 atagagacaa aatagaagaa accgttcata attttctgac caatgaagaa tcatcaacgc
     9061 tatcactttc tgttcacaaa gtatgcgcaa tccacatcgg tatagaatat aatcggggat
     9121 gcctttatct tgaaaaaatg cacccgcagc ttcgctagta atcagtaaac gcgggaagtg
     9181 gagtcaggct ttttttatgg aagagaaaat agacaccaaa gtagccttct tctaacctta
     9241 acggacctac agtgcaaaaa gttatcaaga gactgcatta tagagcgcac aaaggagaaa
     9301 aaaagtaatc taagatgctt tgttagaaaa atagcgctct cgggatgcat ttttgtagaa
     9361 caaaaaagaa gtatagattc tttgttggta aaatagcgct ctcgcgttgc atttctgttc
     9421 tgtaaaaatg cagctcagat tctttgtttg aaaaattagc gctctcgcgt tgcatttttg
     9481 ttttacaaaa atgaagcaca gattcttcgt tggtaaaata gcgctttcgc gttgcatttc
     9541 tgttctgtaa aaatgcagct cagattcttt gtttgaaaaa ttagcgctct cgcgttgcat
     9601 ttttgttcta caaaatgaag cacagatgct tcgttgct
//
