LOCUS       Exported                7182 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Fusion of the pH-sensitive fluorescent biosensor SypHer2 with 
            D-amino acid oxidase; excluded from nucleus. CMV promoter. Can serve
            as a pH control for HyPer..
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-SypHer2-DAAO-NES
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7182)
  AUTHORS   Steinhorn B, Sorrentino A, Badole S, Bogdanova Y, Belousov V, Michel
            T
  TITLE     Chemogenetic generation of hydrogen peroxide in the heart induces 
            severe cardiac dysfunction.
  JOURNAL   Nat Commun. 2018 Oct 2;9(1):4044. doi: 10.1038/s41467-018-06533-2.
  PUBMED    30279532
REFERENCE   2  (bases 1 to 7182)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7182
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     repeat_region   12..141
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        217..520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        521..724
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     675..695
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     721..745
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1329..1338
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1335..2768
                     /codon_start=1
                     /product="improved fluorescent indicator for intracellular 
                     H2O2 (Bilan et al., 2013)"
                     /label=HyPer-3
                     /translation="MEMASQQGETMSGPLHIGLIPTVGPYLLPHIIPMLHQTFPKLEMY
                     LHEAQTHQLLAQLDSGKLDCVILALVKESEAFIEVPLFDEPMLLAIYEDHPWANRECVP
                     MADLAGEKLLMLEDGHSLRDQAMSAGYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQ
                     LADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDEL
                     YNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICT
                     TGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKT
                     RAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNGTGFCFEAGADEDTHFRATSLET
                     LRNMVAVGSGITLLPALAVPPERKRDGVVYLPCIKPEPRRTIGLVYRPGSPLRSRYEQL
                     AEAIRARMDGHFDKVLKQAV"
     primer_bind     1943..1964
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2073..2094)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2334..2353)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3876..3902
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     polyA_signal    3940..4416
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4139..4158)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4456..4596
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4456..4585
                     /label=AAV2 ITR
     rep_origin      4671..5126
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4758..4777)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4968..4989
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5143..5162)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5262..5284
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5322..5340)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5408..5512
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5513..6373
                     /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(5731..5750)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6544..7132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7033..7052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtctagt tattaatagt aatcaattac
      181 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      541 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      601 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      661 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      721 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
      781 tagaagacac cgggaccgat ccagcctccg cggattcgaa tcccggccgg gaacggtgca
      841 ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc
      901 ccacaaaaaa tgctttcttc ttttaatata cttttttgtt tatcttattt ctaatacttt
      961 ccctaatctc tttctttcag ggcaataatg atacaatgta tcatgcctct ttgcaccatt
     1021 ctaaagaata acagtgataa tttctgggtt aaggcaatag caatatttct gcatataaat
     1081 atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata gcagctacaa
     1141 tccagctacc attctgcttt tattttatgg ttgggataag gctggattat tctgagtcca
     1201 agctaggccc ttttgctaat catgttcata cctcttatct tcctcccaca gctcctgggc
     1261 aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag aattgggatt cgaacatcga
     1321 ttgaattcgc caccatggag atggcaagcc agcagggcga gacgatgtcc ggaccgctgc
     1381 acattggttt gattcccaca gttggaccgt acctgctacc gcatattatc cctatgctgc
     1441 accagacctt tccaaagctg gaaatgtatc tgcatgaagc acagacccac cagttactgg
     1501 cgcaactgga cagcggcaaa ctcgattgcg tgatcctcgc gctggtgaaa gagagcgaag
     1561 cattcattga agtgccgttg tttgatgagc caatgttgct ggctatctat gaagatcacc
     1621 cgtgggcgaa ccgcgaatgc gtaccgatgg ccgatctggc aggggaaaaa ctgctgatgc
     1681 tggaagatgg tcactctttg cgcgatcagg caatgtccgc cggctacaac agcgacaacg
     1741 tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
     1801 acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     1861 acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc ctgagcaaag
     1921 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     1981 ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc ggcagcaagg
     2041 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2101 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2161 tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2221 tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag cacgacttct
     2281 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2341 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2401 agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2461 acggcaccgg tttctgtttt gaagccgggg cggatgaaga tacacacttc cgcgcgacca
     2521 gcctggaaac tctgcgcaac atggtggcgg taggtagcgg gatcacttta ctgccagcgc
     2581 tggctgtgcc gccggagcgc aaacgcgatg gggttgttta tctgccgtgc attaagccgg
     2641 aaccacgccg cactattggc ctggtttatc gtcctggctc accgctgcgc agccgctatg
     2701 agcagctggc agaggccatc cgcgcaagaa tggatggcca tttcgataaa gttttaaaac
     2761 aggcggttgg tggtagtggt cacagccaga agagggtggt cgtgctgggc tccggagtca
     2821 tcgggctctc tagcgccctg attctcgcta gaaaaggcta ctccgtgcat atcctggcca
     2881 gagacttgcc tgaggatgtc agctcccaga ccttcgcaag cccctgggcc ggagctaact
     2941 ggacaccatt tatgaccctg acagacggac ccagacaagc caagtgggaa gagtccacct
     3001 tcaagaaatg ggtggagctg gttcccacag gccacgctat gtggctgaaa gggaccagaa
     3061 ggtttgccca gaatgaggat ggcctcctgg gacattggta caaggacatc acccctaact
     3121 ataggccact tccaagctct gaatgtccac ctggagccat tggagtgaca tacgacaccc
     3181 tgagcgtcca cgctcccaag tattgccaat accttgccag agagctgcag aaacttgggg
     3241 ctaccttcga aaggagaaca gtgactagcc tggagcaagc cttcgatgga gctgacctcg
     3301 tggtcaacgc caccgggctg ggagctaagt ccattgccgg catcgatgac caggctgccg
     3361 agcccattag gggacagacc gtgcttgtca aatctccctg taagaggtgc acaatggata
     3421 gctccgaccc tgcttctccc gcttatatca ttcctagacc aggaggagaa gtgatctgtg
     3481 gcggaaccta cggagtcggc gattgggacc tgagcgtgaa tcccgaaaca gtccaaagga
     3541 ttctcaagca ctgcctgaga cttgacccta ccattagctc cgatggaact atcgagggca
     3601 ttgaagtgct gagacataac gttggcctga gacccgccag gagaggagga cctagggtgg
     3661 aggctgaaag aatcgtcttg cccctggacc ggaccaagag ccctctctct ctgggaagag
     3721 ggtccgcacg ggctgcaaaa gagaaggaag tgacccttgt ccacgcctac ggctttagta
     3781 gcgcagggta tcagcaatcc tggggagccg ctgaggacgt ggctcagctg gtcgatgaag
     3841 ccttccaaag ataccacggc gcagctaggg agagcctgcc ccccctggag cggctgaccc
     3901 tgtaagcttg cctcgagcag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     3961 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4021 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4081 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4141 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4201 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4261 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4321 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4381 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4441 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4501 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4561 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4621 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4681 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4741 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4801 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4861 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4921 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4981 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5041 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5101 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5161 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5221 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5281 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5341 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5401 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5461 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5521 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5581 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5641 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5701 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5761 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5821 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5881 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5941 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6001 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6061 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6121 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6181 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6241 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6301 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6361 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6421 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6481 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6541 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6601 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6661 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6721 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6781 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6841 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6901 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6961 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7021 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7081 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7141 caacgcggcc tttttacggt tcctggcctt ttgctggcct tt
//
