LOCUS       Exported                6424 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 6424)
  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..6424
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     primer_bind     165..183
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(221..243)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             477..1280
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     primer_bind     complement(1609..1630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1821..1840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2057..2079
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2071..2088
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2072..2088
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2095..2114
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2095..2113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2116..2139
                     /label=p16NotI_GAP_f
     misc_feature    2122..2203
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     2182..2234
                     /label=p16_GAPprom_f
     primer_bind     complement(2182..2234)
                     /label=GAPprom_p16_r
     primer_bind     complement(2182..2208)
                     /label=uni_p16_r
     primer_bind     2208..2234
                     /label=p16NotI_GAP_f
     promoter        2209..2875
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase"
     primer_bind     2209..2230
                     /label=NotI_GAPprom_f
     primer_bind     complement(2851..2881)
                     /label=mCherry_GAPprom_r
     gene            2900..3616
                     /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     3629..3657
                     /label=ADH1term_mCherry_r
     terminator      3637..3824
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3800..3824)
                     /label=p16SalI_ADH1_r
     primer_bind     complement(3808..3853)
                     /label=p16_ADH1term_r
     primer_bind     complement(3811..3824)
                     /label=SalI_ADH1term_r
     primer_bind     3827..3853
                     /label=uni_p16_f
     primer_bind     3827..3853
                     /label=ADH1term_p16_f
     misc_feature    3827..3831
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     promoter        complement(3843..3861)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3843..3861)
                     /label=T3
                     /note="T3 promoter, forward primer"
     primer_bind     complement(3882..3898)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3882..3898)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3895..3917)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3906..3922
                     /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(3930..3960)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3975..3996
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4113..4130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4284..4872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4364..4383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5043..5903)
                     /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     5666..5685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5904..6008)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    join(6038..6424,1..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 aaggacccta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
      181 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
      241 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
      301 cgtcagcggg tgttggcggc gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      361 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      421 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      481 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      541 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      601 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      661 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      721 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      781 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      841 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      901 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      961 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
     1021 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1081 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1141 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1201 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1261 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1321 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1381 agatgcgtaa ggagaaaata ccgcatcagg cgcggatcct atgcggtgtg aaataccgca
     1441 cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat tttgttaaaa
     1501 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     1561 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     1621 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     1681 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     1741 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     1801 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggct
     1861 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     1921 acagggcgcg tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     1981 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
     2041 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac
     2101 gactcactat agggcgaatt ggagctccac cgcggtggcg gccgctctag aactagtggg
     2161 tcccccgggc tgcaggaatt cgatatcaag cttatcgata ccgtcgactc attatcaata
     2221 ctgccatttc aaagaatacg taaataatta atagtagtga ttttcctaac tttatttagt
     2281 caaaaaatta gccttttaat tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt
     2341 acacagaata tataacatcg taggtgtctg ggtgaacagt ttattcctgg catccactaa
     2401 atataatgga gcccgctttt taagctggca tccagaaaaa aaaagaatcc cagcaccaaa
     2461 atattgtttt cttcaccaac catcagttca taggtccatt ctcttagcgc aactacagag
     2521 aacaggggca caaacaggca aaaaacgggc acaacctcaa tggagtgatg caacctgcct
     2581 ggagtaaatg atgacacaag gcaattgacc cacgcatgta tctatctcat tttcttacac
     2641 cttctattac cttctgctct ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag
     2701 ttccctgaaa ttattcccct acttgactaa taagtatata aagacggtag gtattgattg
     2761 taattctgta aatctatttc ttaaacttct taaattctac ttttatagtt agtctttttt
     2821 ttagttttaa aacaccaaga acttagtttc gaataaacac acataaacaa acaaaggtga
     2881 cggtgctggt ttaattaaca tgtctaaagg tgaagaatta ttcactggtg ttgtcccaat
     2941 tttggttgaa ttagatggtg atgttaatgg tcacaaattt tctgtctccg gtgaaggtga
     3001 aggtgatgct acttacggta aattgacctt aaaatttatt tgtactactg gtaaattgcc
     3061 agttccatgg ccaaccttag tcactacttt aacttggggt gttcaatgtt tttctagata
     3121 cccagatcat atgaaacaac atgacttttt caagtctgcc atgccagaag gttatgttca
     3181 agaaagaact atttttttca aagatgacgg taactacaag accagagctg aagtcaagtt
     3241 tgaaggtgat accttagtta atagaatcga attaaaaggt attgatttta aagaagatgg
     3301 taacatttta ggtcacaaat tggaatacat ttataactct cacaatgttt acatcactgc
     3361 tgacaaacaa aagaatggta tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg
     3421 ttctgttcaa ttagctgacc attatcaaca aaatactcca attggtgatg gtccagtctt
     3481 gttaccagac aaccattact tatccactca atctgcctta tccaaagatc caaacgaaaa
     3541 gagagaccac atggtcttgt tagaatttgt tactgctgct ggtattaccc atggtatgga
     3601 tgaattgtac aaataaggcg cgccacttct aaataagcga atttcttatg atttatgatt
     3661 tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag
     3721 gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct
     3781 caggtatagt atgaggtcgc tcttattgac cacacctcta ccggcagtac cagcttttgt
     3841 tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg
     3901 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3961 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     4021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4081 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4321 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4381 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4441 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4501 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4561 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4621 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4681 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4741 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4801 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4861 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4921 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4981 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5041 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5101 atagttgcct gactgcccgt cgtgtagata actacgatac gggagggctt accatctggc
     5161 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5221 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5281 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5341 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5401 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgaaaaaaa
     5461 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5521 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5581 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5641 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5701 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5761 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5821 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5881 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5941 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6001 gttccgcgca catttccccg aaaagtgcca cctgacggat cgcttgcctg taacttacac
     6061 gcgcctcgta tcttttaatg atggaataat ttgggaattt actctgtatt tatttatttt
     6121 tatgttttgt atttggattt tagaaagtaa ataaagaagg tagaagagtt acggaatgaa
     6181 gaaaaaaaaa taaacaaagg tttaaaaaat ttcaacaaaa agcgtacttt acatatatat
     6241 ttattagaca agaaaagcag attaaataga tatacattcg attaacgata agtaaaatgt
     6301 aaaatcacag gattttcgtg tgtggtcttc tacacagaca agatgaaaca attcggcatt
     6361 aatacctgag agcaggaaga gcaagataaa aggtagtatt tgttggcgat ccccctagag
     6421 tctt
//
