LOCUS       Exported                7505 bp DNA     circular SYN 14-NOV-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7505)
  AUTHORS   The Francis Crick Institute
  TITLE     Direct Submission
  JOURNAL   Exported Sep 8, 2021 from SnapGene Viewer 5.3.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7505
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     164..182
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(220..242)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        566..973
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             974..2068
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(2740..2761)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2952..2971
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3188..3210
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3202..3219
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3203..3219
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3226..3245
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3226..3244
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     3247..3296
                     /label=p16NotI_GAP_f
     misc_feature    3253..3270
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3270..3296
                     /label=p16_GAPprom_f
     promoter        3271..3937
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     3271..3292
                     /label=NotI_GAPprom_f
     primer_bind     3781..3800
                     /label=GPD fw 1
     primer_bind     3828..3847
                     /label=GPD fw 2
     primer_bind     3855..3878
                     /label=p426-sense(GPD_pr)
     primer_bind     complement(3913..3943)
                     /label=mCherry_GAPprom_r
     primer_bind     complement(3938..3950)
                     /label=XhoI_GAPprom_r
     gene            3962..4678
                     /product="Yeast codon optimised (Pombe) enhanced cyan 
                     fluorescent protein (mEx: 435 nm, mEm: 485 nm). From Watson
                     et al, Gene 2008). Complete sequence homology to pAW8-yECFP
                     (http://www.euroscarf.de/plasmid_details.php?accno=P30547) 
                     "
                     /label=eCFP
     primer_bind     4691..4719
                     /label=ADH1term_mCherry_r
     terminator      4699..4886
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     4699..4714
                     /label=NheI_ADHIterm_f
     primer_bind     complement(4862..4911)
                     /label=p16SalI_ADH1_r
     primer_bind     complement(4870..4934)
                     /label=p16_ADH1term_r
     primer_bind     complement(4873..4895)
                     /label=SalI_ADH1term_r
     misc_feature    4891..4912
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4908..4934
                     /label=uni_p16_f
     promoter        complement(4924..4942)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4924..4942)
                     /label=T3
                     /note="T3 promoter, forward primer"
     primer_bind     complement(4963..4979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4963..4979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4976..4998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4987..5003
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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(5011..5041)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5056..5077
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5194..5211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5365..5953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5445..5464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6124..6984)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6747..6766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6985..7089)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(7119..7505,1..117)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac tttctatttt
       61 taatttatat atttatatta aaaaatttaa attataatta tttttatagc acgtgatgaa
      121 aaggacctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
      181 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
      241 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
      301 gtcagcgggt gttggcggcg cgccatcgag gagaacttct agtatatcta catacctaat
      361 attattgcct tattaaaaat ggaatcccaa caattacatc aaaatccaca ttctcttcaa
      421 aatcaattgt cctgtacttc cttgttcatg tgtgttcaaa aacgttatat ttataggata
      481 attatactct atttctcaac aagtaattgg ttgtttggcc gagcggtcta aggcgcctga
      541 ttcaagaaat atcttgaccg cagttaactg tgggaatact caggtatcgt aagatgcaag
      601 agttcgaatc tcttagcaac cattattttt ttcctcaaca taacgagaac acacaggggc
      661 gctatcgcac agaatcaaat tcgatgactg gaaatttttt gttaatttca gaggtcgcct
      721 gacgcatata cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga gagcgccgga
      781 accggctttt catatagaat agagaagcgt tcatgactaa atgcttgcat cacaatactt
      841 gaagttgaca atattattta aggacctatt gttttttcca ataggtggtt agcaatcgtc
      901 ttactttcta acttttctta ccttttacat ttcagcaata tatatatata tatttcaagg
      961 atataccatt ctaatgtctg cccctaagaa gatcgtcgtt ttgccaggtg accacgttgg
     1021 tcaagaaatc acagccgaag ccattaaggt tcttaaagct atttctgatg ttcgttccaa
     1081 tgtcaagttc gatttcgaaa atcatttaat tggtggtgct gctatcgatg ctacaggtgt
     1141 tccacttcca gatgaggcgc tggaagcctc caagaaggct gatgccgttt tgttaggtgc
     1201 tgtgggtggt cctaaatggg gtaccggtag tgttagacct gaacaaggtt tactaaaaat
     1261 ccgtaaagaa cttcaattgt acgccaactt aagaccatgt aactttgcat ccgactctct
     1321 tttagactta tctccaatca agccacaatt tgctaaaggt actgacttcg ttgttgtcag
     1381 agaattagtg ggaggtattt actttggtaa gagaaaggaa gacgatggtg atggtgtcgc
     1441 ttgggatagt gaacaataca ccgttccaga agtgcaaaga atcacaagaa tggccgcttt
     1501 catggcccta caacatgagc caccattgcc tatttggtcc ttggataaag ctaatgtttt
     1561 ggcctcttca agattatgga gaaaaactgt ggaggaaacc atcaagaacg aattccctac
     1621 attgaaggtt caacatcaat tgattgattc tgccgccatg atcctagtta agaacccaac
     1681 ccacctaaat ggtattataa tcaccagcaa catgtttggt gatatcatct ccgatgaagc
     1741 ctccgttatc ccaggttcct tgggtttgtt gccatctgcg tccttggcct ctttgccaga
     1801 caagaacacc gcatttggtt tgtacgaacc atgccacggt tctgctccag atttgccaaa
     1861 gaataaggtc aaccctatcg ccactatctt gtctgctgca atgatgttga aattgtcatt
     1921 gaacttgcct gaagaaggta aggccattga agatgcagtt aaaaaggttt tggatgcagg
     1981 tatcagaact ggtgatttag gtggttccaa cagtaccacc gaagtcggtg atgctgtcgc
     2041 cgaagaagtt aagaaaatcc ttgcttaaaa agattctctt tttttatgat atttgtacat
     2101 aaactttata aatgaaattc ataatagaaa cgacacgaaa ttacaaaatg gaatatgttc
     2161 atagggtaga cgaaactata tacgcaatct acatacattt atcaagaagg agaaaaagga
     2221 ggatgtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga taaggaaaaa
     2281 gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa agttaggtgt
     2341 aacagaaaat catgaaacta tgattcctaa tttatatatt ggaggatttt ctctaaaaaa
     2401 aaaaaaatac aacaaataaa aaacactcaa tgacctgacc atttgatgga gtttaagtca
     2461 ataccttctt gaaccatttc ccataatggt gaaagttccc tcaagaattt tactctgtca
     2521 gaaacggcct tacgacgtag tcgaggatcc tatgcggtgt gaaataccgc acagatgcgt
     2581 aaggagaaaa taccgcatca ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     2641 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2701 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2761 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2821 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2881 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2941 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc tggcaagtgt
     3001 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3061 gtccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     3121 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     3181 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta
     3241 tagggcgaat tggagctcca ccgcggtggc tcattatcaa tactgccatt tcaaagaata
     3301 cgtaaataat taatagtagt gattttccta actttattta gtcaaaaaat tagcctttta
     3361 attctgctgt aacccgtaca tgcccaaaat agggggcggg ttacacagaa tatataacat
     3421 cgtaggtgtc tgggtgaaca gtttattcct ggcatccact aaatataatg gagcccgctt
     3481 tttaagctgg catccagaaa aaaaaagaat cccagcacca aaatattgtt ttcttcacca
     3541 accatcagtt cataggtcca ttctcttagc gcaactacag agaacagggg cacaaacagg
     3601 caaaaaacgg gcacaacctc aatggagtga tgcaacctgc ctggagtaaa tgatgacaca
     3661 aggcaattga cccacgcatg tatctatctc attttcttac accttctatt accttctgct
     3721 ctctctgatt tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga aattattccc
     3781 ctacttgact aataagtata taaagacggt aggtattgat tgtaattctg taaatctatt
     3841 tcttaaactt cttaaattct acttttatag ttagtctttt ttttagtttt aaaacaccaa
     3901 gaacttagtt tcgaataaac acacataaac aaacaaaggt gacggtgctg gtttaattaa
     3961 catgtctaaa ggtgaagaat tattcactgg tgttgtccca attttggttg aattagatgg
     4021 tgatgttaat ggtcacaaat tttctgtctc cggtgaaggt gaaggtgatg ctacttacgg
     4081 taaattgacc ttaaaattta tttgtactac tggtaaattg ccagttccat ggccaacctt
     4141 agtcactact ttaacttggg gtgttcaatg tttttctaga tacccagatc atatgaaaca
     4201 acatgacttt ttcaagtctg ccatgccaga aggttatgtt caagaaagaa ctattttttt
     4261 caaagatgac ggtaactaca agaccagagc tgaagtcaag tttgaaggtg ataccttagt
     4321 taatagaatc gaattaaaag gtattgattt taaagaagat ggtaacattt taggtcacaa
     4381 attggaatac atttataact ctcacaatgt ttacatcact gctgacaaac aaaagaatgg
     4441 tatcaaagct aacttcaaaa ttagacacaa cattgaagat ggttctgttc aattagctga
     4501 ccattatcaa caaaatactc caattggtga tggtccagtc ttgttaccag acaaccatta
     4561 cttatccact caatctgcct tatccaaaga tccaaacgaa aagagagacc acatggtctt
     4621 gttagaattt gttactgctg ctggtattac ccatggtatg gatgaattgt acaaataagg
     4681 cgcgccactt ctaaataagc gaatttctta tgatttatga tttttattat taaataagtt
     4741 ataaaaaaaa taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc
     4801 ttattcttga gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gtatgaggtc
     4861 gctcttattg accacacctc taccggtcga cctggagggg gggcccggta ccagcttttg
     4921 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
     4981 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5041 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5101 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     5161 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5221 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5281 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5341 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5401 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5461 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5521 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5581 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5641 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5701 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5761 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5821 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5881 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5941 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6001 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6061 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6121 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6181 catagttgcc tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6241 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6301 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6361 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6421 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6481 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa
     6541 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6601 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6661 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6721 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6781 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6841 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6901 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6961 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7021 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7081 ggttccgcgc acatttcccc gaaaagtgcc acctgacgga tcgcttgcct gtaacttaca
     7141 cgcgcctcgt atcttttaat gatggaataa tttgggaatt tactctgtat ttatttattt
     7201 ttatgttttg tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga
     7261 agaaaaaaaa ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata
     7321 tttattagac aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg
     7381 taaaatcaca ggattttcgt gtgtggtctt ctacacagac aagatgaaac aattcggcat
     7441 taatacctga gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga
     7501 gtctt
//
