LOCUS       Exported                8004 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  The genetically encoded fluorescent H2O2 sensor roGFP2-Tsa2-Delta-CR
            cloned in the yeast p415 vector under the control of a TEF promoter.
            For cytosolic expression..
ACCESSION   .
VERSION     .
KEYWORDS    p415 TEF roGFP2‐Tsa2-Delta-CR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8004)
  AUTHORS   Morgan B, Van Laer K, Owusu TN, Ezerina D, Pastor-Flores D, Amponsah
            PS, Tursch A, Dick TP
  TITLE     Real-time monitoring of basal H2O2 levels with peroxiredoxin-based 
            probes.
  JOURNAL   Nat Chem Biol. 2016 Jun;12(6):437-43. doi: 10.1038/nchembio.2067. 
            Epub 2016 Apr 18.
  PUBMED    27089028
REFERENCE   2  (bases 1 to 8004)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8004
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             715..1575
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2658..2688
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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)."
     primer_bind     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2793..3191
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     terminator      4632..4879
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4648..4666)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4897..4916)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4898..4916)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4926..4943)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4926..4942)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4935..4957)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5083..5538
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5170..5189)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5380..5401
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5839..6246
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             6247..7341
                     /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(7835..7854)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7954..7976
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctcatagctt caaaatgttt ctactccttt
     2821 tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa aacacccaag
     2881 cacagcatac taaatttccc ctctttcttc ctctagggtg tcgttaatta cccgtactaa
     2941 aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa aaaggcaata
     3001 aaaattttta tcacgtttct ttttcttgaa aatttttttt ttgatttttt tctctttcga
     3061 tgacctccca ttgatattta agttaataaa cggtcttcaa tttctcaagt ttcagtttca
     3121 tttttcttgt tctattacaa ctttttttac ttcttgctca ttagaaagaa agcatagcaa
     3181 tctaatctaa gttttctaga ggatccacca tggctagcga attctcaaag ggtgaagaat
     3241 tgtttacagg tgttgttcct attttagtcg aattggacgg tgacgttaat ggtcataagt
     3301 ttagtgttag tggtgaaggt gaaggtgacg caacatacgg taaattgacc ttgaagttta
     3361 tttcaactac tggtaaattg ccagttcctt ggccaacttt ggtaaccact ttaacatatg
     3421 gtgtacaatg tttcagtaga taccctgatc atatgaaaca acacgacttt ttcaagtctg
     3481 ctatgccaga aggttacgtt caagaaagaa ctattttctt taaggatgac ggtaactaca
     3541 agaccagagc agaagtcaaa tttgaaggtg acactttggt taacagaatc gaattgaagg
     3601 gtatcgattt caaggaagac ggtaacatct tgggtcataa attggaatac aactacaact
     3661 gtcacaatgt atacataatg gccgataagc aaaagaatgg tatcaaagtc aacttcaaga
     3721 tcagacataa catcgaagat ggttctgttc aattagctga ccactatcaa caaaatacac
     3781 ctattggtga cggtcctgtt ttgttaccag acaaccatta cttgtctacc tgctcagctt
     3841 tatccaaaga tccaaatgaa aagagagacc atatggtatt gttagaattt gtcaccgcag
     3901 caggtatcac attgggtatg gatgaattgt ataaaactag tggtggttca ggtggtggtg
     3961 gttcaggtgg tggtggttca ggtggaggag gatcaggagg aggaggatca ggaggaggag
     4021 gatcaggagg agaattcgta gcagaagttc aaaaacaagc cccaccattt aagaaaaccg
     4081 ccgtagtcga cggtatcttc gaggaaattt cactggaaaa gtataaaggt aagtacgttg
     4141 ttctagcttt tgtcccattg gctttttcat ttgtctgtcc aactgagatt gttgcgtttt
     4201 ccgatgccgc caagaaattc gaagatcagg gcgcccaagt tttatttgcc tccaccgact
     4261 ctgaatattc cttactggca tggaccaacc ttcccagaaa agacggtgga ttaggtccag
     4321 ttaaagttcc tttgcttgct gataagaatc attccttatc cagagactat ggcgttttga
     4381 ttgaaaaaga aggtatagct ttaagaggtt tgttcataat cgacccgaag ggaatcatta
     4441 gacatatcac tatcaatgat ttatctgttg gcagaaacgt caatgaagct ttgagattag
     4501 tcgaaggttt ccagtggact gacaaaaatg gtacagtttt gccagccaac tggaccccag
     4561 gagccgccac catcaaacct gacgttaaag attccaagga gtatttcaaa aatgccaata
     4621 attaactcga gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc
     4681 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     4741 tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
     4801 cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     4861 ctcgaaggct ttaatttgcg gccggtaccc aattcgccct atagtgagtc gtattacgcg
     4921 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     4981 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     5041 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg
     5101 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     5161 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5221 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5281 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5341 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5401 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5461 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5521 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5581 acaccgcata tcgaccggtc gaggagaact tctagtatat ctacatacct aatattattg
     5641 ccttattaaa aatggaatcc caacaattac atcaaaatcc acattctctt caaaatcaat
     5701 tgtcctgtac ttccttgttc atgtgtgttc aaaaacgtta tatttatagg ataattatac
     5761 tctatttctc aacaagtaat tggttgtttg gccgagcggt ctaaggcgcc tgattcaaga
     5821 aatatcttga ccgcagttaa ctgtgggaat actcaggtat cgtaagatgc aagagttcga
     5881 atctcttagc aaccattatt tttttcctca acataacgag aacacacagg ggcgctatcg
     5941 cacagaatca aattcgatga ctggaaattt tttgttaatt tcagaggtcg cctgacgcat
     6001 ataccttttt caactgaaaa attgggagaa aaaggaaagg tgagagcgcc ggaaccggct
     6061 tttcatatag aatagagaag cgttcatgac taaatgcttg catcacaata cttgaagttg
     6121 acaatattat ttaaggacct attgtttttt ccaataggtg gttagcaatc gtcttacttt
     6181 ctaacttttc ttacctttta catttcagca atatatatat atatatttca aggatatacc
     6241 attctaatgt ctgcccctaa gaagatcgtc gttttgccag gtgaccacgt tggtcaagaa
     6301 atcacagccg aagccattaa ggttcttaaa gctatttctg atgttcgttc caatgtcaag
     6361 ttcgatttcg aaaatcattt aattggtggt gctgctatcg atgctacagg tgttccactt
     6421 ccagatgagg cgctggaagc ctccaagaag gctgatgccg ttttgttagg tgctgtgggt
     6481 ggtcctaaat ggggtaccgg tagtgttaga cctgaacaag gtttactaaa aatccgtaaa
     6541 gaacttcaat tgtacgccaa cttaagacca tgtaactttg catccgactc tcttttagac
     6601 ttatctccaa tcaagccaca atttgctaaa ggtactgact tcgttgttgt cagagaatta
     6661 gtgggaggta tttactttgg taagagaaag gaagacgatg gtgatggtgt cgcttgggat
     6721 agtgaacaat acaccgttcc agaagtgcaa agaatcacaa gaatggccgc tttcatggcc
     6781 ctacaacatg agccaccatt gcctatttgg tccttggata aagctaatgt tttggcctct
     6841 tcaagattat ggagaaaaac tgtggaggaa accatcaaga acgaattccc tacattgaag
     6901 gttcaacatc aattgattga ttctgccgcc atgatcctag ttaagaaccc aacccaccta
     6961 aatggtatta taatcaccag caacatgttt ggtgatatca tctccgatga agcctccgtt
     7021 atcccaggtt ccttgggttt gttgccatct gcgtccttgg cctctttgcc agacaagaac
     7081 accgcatttg gtttgtacga accatgccac ggttctgctc cagatttgcc aaagaataag
     7141 gtcaacccta tcgccactat cttgtctgct gcaatgatgt tgaaattgtc attgaacttg
     7201 cctgaagaag gtaaggccat tgaagatgca gttaaaaagg ttttggatgc aggtatcaga
     7261 actggtgatt taggtggttc caacagtacc accgaagtcg gtgatgctgt cgccgaagaa
     7321 gttaagaaaa tccttgctta aaaagattct ctttttttat gatatttgta cataaacttt
     7381 ataaatgaaa ttcataatag aaacgacacg aaattacaaa atggaatatg ttcatagggt
     7441 agacgaaact atatacgcaa tctacataca tttatcaaga aggagaaaaa ggaggatgta
     7501 aaggaataca ggtaagcaaa ttgatactaa tggctcaacg tgataaggaa aaagaattgc
     7561 actttaacat taatattgac aaggaggagg gcaccacaca aaaagttagg tgtaacagaa
     7621 aatcatgaaa ctatgattcc taatttatat attggaggat tttctctaaa aaaaaaaaaa
     7681 tacaacaaat aaaaaacact caatgacctg accatttgat ggagtttaag tcaatacctt
     7741 cttgaaccat ttcccataat ggtgaaagtt ccctcaagaa ttttactctg tcagaaacgg
     7801 ccttaacgac gtagtcgata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     7861 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     7921 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7981 caccgtcatc accgaaacgc gcga
//
