LOCUS       sequence-247763-        8222 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mitochondrial matrix targeted super ecliptic pHluorin with C 
            terminal mRuby2 fusion tag in yeast expression vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8222)
  AUTHORS   Dodd BJT, Kralj JM
  TITLE     Live Cell Imaging Reveals pH Oscillations in Saccharomyces 
            cerevisiae During Metabolic Transitions.
  JOURNAL   Sci Rep. 2017 Oct 24;7(1):13922. doi: 10.1038/s41598-017-14382-0.
  PUBMED    29066766
REFERENCE   2  (bases 1 to 8222)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2017-10-24"; volume: "7(1):13922. doi"; pages: " 
            10.1038/s41598-017-14382-0"
COMMENT     Sequence Label: pKL07
FEATURES             Location/Qualifiers
     source          1..8222
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(11..29)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    71..574
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        611..715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(716..784,785..1576)
                     /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(934..953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1747..2335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2236..2255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2489..2506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2623..2644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2659..2664,2665..2682,2683..2689)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2697..2713
                     /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     2702..2724
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2721..2737
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2721..2737
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2756..2776
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2758..2776
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2794..3192
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     terminator      4853..5100
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4869..4887)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(5118..5137)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5119..5137)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5147..5164)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5147..5163)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5156..5178)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5304..5759
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5391..5410)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5601..5622
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6060..6465
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             6466..7560
                     /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(8054..8073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8173..8195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
       61 tcttaggacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
      121 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
      181 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
      241 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
      301 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
      361 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
      421 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
      481 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
      541 aatttaaatt ataattattt ttatagcacg tgatgaaaag gacccaggtg gcacttttcg
      601 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
      661 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
      721 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
      781 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
      841 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
      901 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
      961 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1021 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1081 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     1141 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     1201 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     1261 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     1321 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     1381 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     1441 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     1501 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     1561 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     1621 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     1681 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     1741 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     1801 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     1861 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     1921 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     1981 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2041 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2101 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     2161 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     2221 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     2281 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     2341 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     2401 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     2461 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     2521 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     2581 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     2641 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     2701 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
     2761 taaccctcac taaagggaac aaaagctgga gctcatagct tcaaaatgtt tctactcctt
     2821 ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca aaacacccaa
     2881 gcacagcata ctaaatttcc cctctttctt cctctagggt gtcgttaatt acccgtacta
     2941 aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat
     3001 aaaaattttt atcacgtttc tttttcttga aaattttttt tttgattttt ttctctttcg
     3061 atgacctccc attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc
     3121 atttttcttg ttctattaca acttttttta cttcttgctc attagaaaga aagcatagca
     3181 atctaatcta agttttctag agatggcctc cactcgtgtc ctcgcctctc gcctggcctc
     3241 ccagatggct gcttccgcca aggttgcccg ccctgctgtc cgcgttgctc aggtcagcaa
     3301 gcgcaccatc cagactggct cccccctcca gaccctcaag cgcacccaga tgacctccat
     3361 cgtcaacgcc accacccgcc aggctttcca gaagcgcgcc tactcttccg ctggatccat
     3421 gtctaagggc gaggaactgt tcactggtgt ggttccaatc ctggttgagc tggacggcga
     3481 tgtaaacggt cacaagttca gcgttagcgg cgaaggtgag ggcgacgcta cctacggtaa
     3541 actgactctg aagttcatct gcaccaccgg taaactgcct gttccgtggc caactctggt
     3601 caccaccttc agctacggtg ttcagtgtct gacccgctac cctgatcaca tgaaacgcca
     3661 tgacttcttc aaaagcgcga tgccggaagg ctatgttcaa gaacgtacca tctttttcaa
     3721 ggatgacggc aattataaaa ctcgtgccga agtaaagttc gaaggcgaca ccctggttaa
     3781 ccgtattgaa ctgaaaggta ttgatttcaa agaagatggt aacattctgg gtcacaaact
     3841 ggaatataac tacaacgacc accaggttta catcatggcg gataaacaga aaaacggcat
     3901 caaagctaac ttcaaaatcc gtcataacat cgaagacggc ggtgtgcagc tggctgacca
     3961 ctaccagcag aacaccccga tcggtgatgg tccggttctg ctgccggata atcactacct
     4021 gttcaccacg tctactctgt ccaaagaccc gaacgaaaaa cgtgatcaca tggtcctgct
     4081 ggaattcgtt actgcagcag gtatcaccca cggcatggat gaactgtaca agatggtttc
     4141 aaagggtgaa gaacttatta aagaaaacat gagaatgaag gtagttatgg aaggaagcgt
     4201 taatggccat caattcaagt gtacgggaga aggagagggt aggccttacg aagggacgca
     4261 aactatgagg attaaggtta ttgagggtgg tccattgcca ttcgcattcg acatcttagc
     4321 tacatctttt atgtatggaa gtcgtacgtt cataaagtat cctgctgata ttccagactt
     4381 ctttaaacag agcttccctg agggctttac atgggaaaga gtgaccagat atgaagatgg
     4441 tggtgttgtt actgttactc aagacacttc tttagaagac ggtgaactag tttataatgt
     4501 aaaagttagg ggtgttaact tcccgtctaa cgggccagtc atgcaaaaga agacaaaagg
     4561 atgggaacct aacacggaga tgatgtatcc cgctgacggt ggtttgagag gctatacgga
     4621 tattgcgttg aaagtcgacg gaggcggtca tttgcattgc aattttgtta caacttacag
     4681 gagtaagaag acagtaggta atatcaagat gcccggcgtt cacgcagttg accatcgttt
     4741 ggaacgtatt gaggaatcag ataatgagac atacgtggta cagagggagg ttgccgtagc
     4801 taagtacagt aacttggggg gtgggatgga cgaattatat aaataactcg agtcatgtaa
     4861 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     4921 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     4981 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     5041 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     5101 ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     5161 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5221 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5281 tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     5341 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     5401 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5461 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     5521 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5581 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5641 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5701 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc
     5761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atcgaccggt
     5821 cgaggagaac ttctagtata tctacatacc taatattatt gccttattaa aaatggaatc
     5881 ccaacaatta catcaaaatc cacattctct tcaaaatcaa ttgtcctgta cttccttgtt
     5941 catgtgtgtt caaaaacgtt atatttatag gataattata ctctatttct caacaagtaa
     6001 ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag aaatatcttg accgcagtta
     6061 actgtgggaa tactcaggta tcgtaagatg caagagttcg aatctcttag caaccattat
     6121 ttttttcctc aacataacga gaacacacag gggcgctatc gcacagaatc aaattcgatg
     6181 actggaaatt ttttgttaat ttcagaggtc gcctgacgca tatacctttt tcaactgaaa
     6241 aattgggaga aaaaggaaag gtgagagcgc cggaaccggc ttttcatata gaatagagaa
     6301 gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc
     6361 tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt
     6421 acatttcagc aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctaag
     6481 aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga agccattaag
     6541 gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga aaatcattta
     6601 attggtggtg ctgctatcga tgctacaggt gttccacttc cagatgaggc gctggaagcc
     6661 tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg gtcctaaatg gggtaccggt
     6721 agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt gtacgccaac
     6781 ttaagaccat gtaactttgc atccgactct cttttagact tatctccaat caagccacaa
     6841 tttgctaaag gtactgactt cgttgttgtc agagaattag tgggaggtat ttactttggt
     6901 aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata caccgttcca
     6961 gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga gccaccattg
     7021 cctatttggt ccttggataa agctaatgtt ttggcctctt caagattatg gagaaaaact
     7081 gtggaggaaa ccatcaagaa cgaattccct acattgaagg ttcaacatca attgattgat
     7141 tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat aatcaccagc
     7201 aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggttc cttgggtttg
     7261 ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg tttgtacgaa
     7321 ccatgccacg gttctgctcc agatttgcca aagaataagg tcaaccctat cgccactatc
     7381 ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg taaggccatt
     7441 gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt aggtggttcc
     7501 aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat ccttgcttaa
     7561 aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat tcataataga
     7621 aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta tatacgcaat
     7681 ctacatacat ttatcaagaa ggagaaaaag gaggatgtaa aggaatacag gtaagcaaat
     7741 tgatactaat ggctcaacgt gataaggaaa aagaattgca ctttaacatt aatattgaca
     7801 aggaggaggg caccacacaa aaagttaggt gtaacagaaa atcatgaaac tatgattcct
     7861 aatttatata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata aaaaacactc
     7921 aatgacctga ccatttgatg gagtttaagt caataccttc ttgaaccatt tcccataatg
     7981 gtgaaagttc cctcaagaat tttactctgt cagaaacggc cttaacgacg tagtcgatat
     8041 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     8101 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     8161 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     8221 cg
//
