LOCUS       Exported                9040 bp ds-DNA     circular SYN 06-SEP-2022
DEFINITION  a biosensor comprised of a fast-maturing pH-stable red fluorescent 
            protein fused to a pH-sensitive green fluorescent protein variant. 
            Cytosolic localization..
ACCESSION   .
VERSION     .
KEYWORDS    pAS1NB c Rosella I
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9040)
  AUTHORS   Rosado CJ, Mijaljica D, Hatzinisiriou I, Prescott M, Devenish RJ
  TITLE     Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover 
            of cytosol and organelles in yeast.
  JOURNAL   Autophagy. 2008 Feb;4(2):205-13. Epub 2007 Nov 21.
  PUBMED    18094608
REFERENCE   2  (bases 1 to 9040)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 6, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9040
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        142..172
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     185..207
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     204..220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    1656..1708
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1718..1727
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1724..2398
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGRFIYKVKFIGVNFPSDGPVMQKKTMGWEPSTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERTEGRHH
                     LFL"
     primer_bind     complement(1854..1872)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1904..1924)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     2122..2141
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2316..2339
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             2429..3136
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKANF
                     KIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEFV
                     TAAGITQGMDELYK"
     primer_bind     complement(2453..2481)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3076..3096
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3252..3525
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     misc_feature    3532..3588
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3589..3606)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3589..3605)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3598..3620)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4042..5388
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     primer_bind     complement(7048..7066)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7134..7238
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7239..8099
                     /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(7457..7476)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8270..8858
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8759..8778
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9012..9029
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
       61 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttcg
      241 aattctgcag tcgactctag agctgtagaa ttttctcatt ggtcacatta caacctgaaa
      301 atactttatc tacaatcata ccattcttat aacatgtccc cttaatacta ggatcaggca
      361 tgaacgcatc acagacaaaa tcttcttgac aaacgtcaca attgatccct ccccatccgt
      421 tatcacaatg acaggtgtca ttttgtgctc ttatgggacg atccttatta ccgctttcat
      481 ccggtgatag accgccacag aggggcagag agcaatcatc acctgcaaac ccttctatac
      541 actcacatct accagtgtac gaattgcatt cagaaaactg tttgcattca aaaataggta
      601 gcatacaatt aaaacatggc gggcacgtat cattgccctt atcttgtgca gttagacgcg
      661 aatttttcga agaagtacct tcaaagaatg gggtctcatc ttgtttggaa gtaccactga
      721 gcaggataat aatagaaatg ataatatact atagtagaga taacgtcgat gacttcccat
      781 actgtaattg cttttagttg tgtattttta gtgtgcaagt ttctgtaaat cgattaattt
      841 ttttttcttt cctcttttta ttaaccttaa tttttatttt agattcctga cttcaactca
      901 agacgcacag atattataac atctgcacaa taggcatttg caagaattac tcgtgagtaa
      961 ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc
     1021 tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc
     1081 ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg
     1141 tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc
     1201 tctcttccta ttgattgcag cttccaattt cgtcacacaa caaggtccta gcgacggctc
     1261 acaggttttg taacaagcaa tcgaaggttc tggaatggcg ggaaagggtt tactaccaca
     1321 tgctatgatg cccactgtga tctccagagc aaagttcgtt cgatcgtact gttactctct
     1381 ctctttcaaa cagaattgtc cgaatcgtgt gacaacaaca gcctgttctc acacactctt
     1441 ttcttctaac caagggggtg gtttagttta gtagaacctc gtgaaactta catttacata
     1501 tatataaact tgcataaatt ggtcaatgca agaaatacat atttggtctt ttctaattcg
     1561 tagtttttca agttcttaga tgctttcttt ttctcttttt tacagatcat caaggaagta
     1621 attatctact ttttctctag aagatctaaa catgtctcga gctcaagctt cgaattctgc
     1681 agtcgacggt accgcgggcc cgggatcccc actagtcgcc accatggcct cctccgagga
     1741 cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag ggctccgtga acggccacga
     1801 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     1861 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
     1921 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
     1981 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     2041 cgtgacccag gactcctccc tgcaggacgg ccgcttcatc tacaaggtga agttcatcgg
     2101 cgtgaacttc ccctccgacg gccccgtaat gcagaagaag actatgggct gggagccctc
     2161 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggccctgaa
     2221 gctgaaggac ggcggccact acctggtgga gttcaagtcc atctacatgg ccaagaagcc
     2281 cgtgcagctg cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga
     2341 ggactacacc atcgtggagc agtacgagcg caccgagggc cgccaccacc tgttcctgtc
     2401 tgatccctgg tcttcttcgg gagatctaaa aggagaagaa cttttcactg gagttgtccc
     2461 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
     2521 tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact
     2581 acctgttcct tggccaacac ttgtcactac tttaacttat ggtgttcaat gcttttcaag
     2641 atacccagat catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt
     2701 acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa
     2761 gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga
     2821 tggaaacatt cttggacaca aattggaata caactataac gatcaccagg tgtacatcat
     2881 ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attagacaca acattgaaga
     2941 tggaggcgtt caactagcag accattatca acaaaatact ccaattggcg atgggcccgt
     3001 ccttttacca gacaaccatt acctgtttac aacttctact ctttcgaaag atcccaacga
     3061 aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacaaggcat
     3121 ggatgaacta tacaaatctg atcactgagc ggccgcggat ctcccatgtc tctactggtg
     3181 gtggtgcttc tttggaatta ttggaaggta aggaattgcc aggtgttgct ttcttatccg
     3241 aaaagaaata aattgaattg aattgaaatc gatagatcaa tttttttctt ttctctttcc
     3301 ccatccttta cgctaaaata atagtttatt ttattttttg aatatttttt atttatatac
     3361 gtatatatag actattatta tcttttaata gattattaag atttttatta aaaaaaaatt
     3421 cgtccctctt tttaatgcct tttatgcagt ttttttttcc cattcgatat ttctatgttc
     3481 gggtttcagc gtattttaag tttaataact cgaaaattct gcgtttcgaa aaagcttgca
     3541 tgcctgcagg tcgactctag agaatccccg ggtaccgagc tcgaattcac tggccgtcgt
     3601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     3661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3721 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg
     3781 cggtatttca caccgcatat atcggatcgt acttgttacc catcattgaa ttttgaacat
     3841 ccgaacctgg gagttttccc tgaaacagat agtatatttg aacctgtata ataatatata
     3901 gtctagcgct ttacggaaga caatgtatgt atttcggttc ctggagaaac tattgcatct
     3961 attgcatagg taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt ccatcttgca
     4021 cttcaatagc atatctttgt taacgaagca tctgtgcttc attttgtaga acaaaaatgc
     4081 aacgcgagag cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa
     4141 tgcaacgcga aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa
     4201 aatgcaacgc gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac
     4261 agaaatgcaa cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct
     4321 acaaaaatgc atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc
     4381 tcctttgtgc gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag
     4441 gttagaagaa ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact
     4501 tcccgcgttt actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc
     4561 ccgattatat tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt
     4621 tgatgattct tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac
     4681 tacgtatagg aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt
     4741 actacaattt ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg
     4801 agtttagatg caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca
     4861 cagagatata tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata
     4921 ttttagtagc tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc
     4981 gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt ctagctagag aataggaact
     5041 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc
     5101 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata
     5161 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct
     5221 atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg
     5281 gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat
     5341 tggattagtc tcatccttca atgctatcat ttcctttgat attggatcga tccgatgata
     5401 agctgtcaaa catgagaatt aattctaccc tatgaacata ttccattttg taatttcgtg
     5461 tcgtttctat tatgaatttc atttataaag tttatgtaca aatatcataa aaaaagagaa
     5521 tctttttaag caaggatttt cttaacttct tcggcgacag catcaccgac ttcggtggta
     5581 ctgttggaac cacctaaatc accagttctg atacctgcat ccaaaacctt tttaactgca
     5641 tcttcaatgg ccttaccttc ttcaggcaag ttcaatgaca atttcaacat cattgcagca
     5701 gacaagatag tggcgatagg gtcaacctta ttctttggca aatctggagc agaaccgtgg
     5761 catggttcgt acaaaccaaa tgcggtgttc ttgtctggca aagaggccaa ggacgcagat
     5821 ggcaacaaac ccaaggaacc tgggataacg gaggcttcat cggagatgat atcaccaaac
     5881 atgttgctgg tgattataat accatttagg tgggttgggt tcttaactag gatcatggcg
     5941 gcagaatcaa tcaattgatg ttgaaccttc aatgtaggaa attcgttctt gatggtttcc
     6001 tccacagttt ttctccataa tcttgaagag gccaaaacat tagctttatc caaggaccaa
     6061 ataggcaatg gtggctcatg ttgtagggcc atgaaagcgg ccattcttgt gattctttgc
     6121 acttctggaa cggtgtattg ttcactatcc caagcgacac catcaccatc gtcttccttt
     6181 ctcttaccaa agtaaatacc tcccactaat tctctgacaa caacgaagtc agtaccttta
     6241 gcaaattgtg gcttgattgg agataagtct aaaagagagt cggatgcaaa gttacatggt
     6301 cttaagttgg cgtacaattg aagttcttta cggattttta gtaaaccttg ttcaggtcta
     6361 acactacctg taccccattt aggaccaccc acagcaccta acaaaacggc atcaaccttc
     6421 ttggaggctt ccagcgcctc atctggaagt gggacacctg tagcatcgat agcagcacca
     6481 ccaattaaat gattttcgaa atcgaacttg acattggaac gaacatcaga aatagcttta
     6541 agaaccttaa tggcttcggc tgtgatcttg accaacgtgg tcacctggca aaacgacgat
     6601 cttcttaggg gcagacatag gggcagacat tagaatgtat atccttgaaa tatatatata
     6661 tattgctgaa atgtaaaagg taagaaaagt tagaataaga cgattgctaa ccacctattg
     6721 gaaaaaacaa taggtcctta aataatattg tcaacttcaa gtattgtgat gcaagcattt
     6781 agtcatgaac gcttctctat tctatatgaa aagccggttc cggcctctca cctttccttt
     6841 ttctcccaat ttttcagttg aaaaaggtat atgcgcggcg acctctgaaa ttaacaaaaa
     6901 atttccagtc atcgaatttg tctgtgcgat agcgcccctg tgtgttctcg ttatgttgag
     6961 gaaaaaaata atggttgcta agagattcga actcttgcat cttacgatac ctgagtattc
     7021 ccacagttaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     7081 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     7141 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     7201 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     7261 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     7321 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     7381 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     7441 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     7501 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     7561 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     7621 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     7681 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7741 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7801 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7861 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7921 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7981 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     8041 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     8101 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     8161 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     8221 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     8281 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     8341 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     8401 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     8461 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     8521 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     8581 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     8641 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     8701 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     8761 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8821 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8881 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     8941 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     9001 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
//
