LOCUS       Exported                8265 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expresses ratiometric sensor of lysosomal lumenal pH.
ACCESSION   .
VERSION     .
KEYWORDS    pCAG R2pH-LAMP1-3xFLAG
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8265)
  AUTHORS   Ponsford AH, Ryan TA, Raimondi A, Cocucci E, Wycislo SA, Frohlich F,
            Swan LE, Stagi M
  TITLE     Live imaging of intra-lysosome pH in cell lines and primary neuronal
            culture using a novel genetically encoded biosensor.
  JOURNAL   Autophagy. 2020 Jun 9:1-19. doi: 10.1080/15548627.2020.1771858.
  PUBMED    32515674
REFERENCE   2  (bases 1 to 8265)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8265
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..657
                     /label=chicken beta-actin promoter
     intron          658..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1730..1739
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1813..2520
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             2533..3240
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             3262..3975
                     /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="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(3292..3320)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3915..3935
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             5131..5196
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5283..5302)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5348..5403
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5402..5421)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5764..5780)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5764..5780)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5777..5799)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5788..5804
                     /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(5812..5842)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5857..5878
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5937..6132
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5983..6118
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6045..6064
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    6138..6272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6175..6194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6229..6248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6340..6357)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6511..7099)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6591..6610)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7270..8130)
                     /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     7893..7912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8131..8235)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgcca ggcggggcgg ggcggggcga ggggcggggc
      541 ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct
      601 tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga
      661 gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc
      841 cttgaggggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcac catggcggcc
     1741 cccggcgccc ggcggccgct gctcctgctg ctgctggcag gccttgcaca tggcgcctca
     1801 gcagctagcg gtatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     1861 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     1921 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     1981 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     2041 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2101 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2161 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     2221 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     2281 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     2341 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     2401 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2461 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2521 ggagctagcg gtatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     2581 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     2641 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     2701 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     2761 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
     2821 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2881 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     2941 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     3001 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     3061 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     3121 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     3181 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     3241 ggagctagcg gggggcccgg gatgagtaaa ggagaagaac ttttcactgg agttgtccca
     3301 attcttgttg aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt
     3361 gaaggtgatg caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta
     3421 cctgttcctt ggccaacact tgtcactact ttaacttatg gcgttcaatg cttttcaaga
     3481 tacccagatc atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta
     3541 caggaaagaa ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag
     3601 tttgaaggtg atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat
     3661 ggaaacattc ttggacacaa attggaatac aactataacg atcaccaggt gtacatcatg
     3721 gcagacaaac aaaagaatgg aatcaaagct aacttcaaaa ttagacacaa cattgaagat
     3781 ggaggcgttc aactagcaga ccattatcaa caaaatactc caattggcga tgggcccgtc
     3841 cttttaccag acaaccatta cctgtttaca acttctactc tttcgaaaga tcccaacgaa
     3901 aagagagacc acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg
     3961 gatgaactat acaaacccgg ggggctcttt gaggtgaaaa acaatggcac aacgtgtata
     4021 atggccagct tctctgcctc ctttctgacc acctacgaga ctgcgaatgg ttctcagatc
     4081 gtgaacattt ccctgccagc ctctgcagaa gtactgaaaa atggcagttc ttgtggtaaa
     4141 gaaaatgttt ctgaccccag cctcacaatt acttttggaa gaggatattt actgacactc
     4201 aacttcacaa aaaatacaac acgttacagt gtccagcata tgtattttac atataacttg
     4261 tcagatacag aacattttcc caatgccatc agcaaagaga tctacaccat ggattccaca
     4321 actgacatca aggcagacat caacaaagca taccggtgtg tcagtgatat ccgggtctac
     4381 atgaagaatg tgaccgttgt gctccgggat gccactatcc aggcctacct gtcgagtggc
     4441 aacttcagca aggaagagac acactgcaca caggatggac cttccccaac cactgggcca
     4501 cccagcccct caccaccact tgtgcccaca aaccccactg tatccaagta caatgttact
     4561 ggtaacaacg gaacctgcct gctggcctct atggcactgc aactgaatat cacctacctg
     4621 aaaaaggaca acaagacggt gaccagagcg ttcaacatca gcccaaatga cacatctagt
     4681 gggagttgcg gtatcaactt ggtgaccctg aaagtggaga acaagaacag agccctggaa
     4741 ttgcagtttg ggatgaatgc cagctctagc ctgtttttcc tgcaaggagt gcgcttgaat
     4801 atgactcttc ctgatgccct agtgcccaca ttcagcatct ccaaccattc actgaaagct
     4861 cttcaggcca ctgtgggaaa ctcatacaag tgcaacactg aggaacacat ctttgtcagc
     4921 aagatgctct ccctcaatgt cttcagtgtg caggtccagg ctttcaaggt ggacagtgac
     4981 aggtttgggt ctgtggaaga gtgtgttcag gatggtaaca acatgttgat ccccattgct
     5041 gtgggcggtg ccctggcagg gctggtcctc atcgtcctca ttgcctacct cattggcagg
     5101 aagaggagtc acgccggcta tcagaccatc gactacaaag accatgacgg tgattataaa
     5161 gatcatgaca tcgactacaa ggatgacgat gacaagtagg aattcactcc tcaggtgcag
     5221 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     5281 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     5341 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     5401 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     5461 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     5521 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     5581 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     5641 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     5701 atagctgtcc ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc
     5761 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5821 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5881 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     5941 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     6001 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     6061 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     6121 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6181 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6241 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     6301 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6361 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6421 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6481 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6541 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6601 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6661 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6721 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6781 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6841 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6901 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6961 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     7021 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     7081 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     7141 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     7201 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7261 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7321 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7381 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7441 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7501 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7561 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7621 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7681 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7741 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7801 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7861 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7921 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7981 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     8041 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     8101 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     8161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     8221 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtc
//
