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   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        206..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..713
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     664..684
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     710..734
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1318..1327
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1324..2757
                     /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     1932..1953
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2062..2083)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2323..2342)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3865..3891
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     polyA_signal    3929..4405
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4128..4147)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4445..4585
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4445..4574
                     /label=AAV2 ITR
     rep_origin      4660..5115
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4747..4766)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4957..4978
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5132..5151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5251..5273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5311..5329)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5397..5501
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5502..6362
                     /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(5720..5739)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6533..7121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7022..7041
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      541 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      601 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      661 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag
      721 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc
      781 gggaccgatc cagcctccgc ggattcgaat cccggccggg aacggtgcat tggaacgcgg
      841 attccccgtg ccaagagtga cgtaagtacc gcctatagag tctataggcc cacaaaaaat
      901 gctttcttct tttaatatac ttttttgttt atcttatttc taatactttc cctaatctct
      961 ttctttcagg gcaataatga tacaatgtat catgcctctt tgcaccattc taaagaataa
     1021 cagtgataat ttctgggtta aggcaatagc aatatttctg catataaata tttctgcata
     1081 taaattgtaa ctgatgtaag aggtttcata ttgctaatag cagctacaat ccagctacca
     1141 ttctgctttt attttatggt tgggataagg ctggattatt ctgagtccaa gctaggccct
     1201 tttgctaatc atgttcatac ctcttatctt cctcccacag ctcctgggca acgtgctggt
     1261 ctgtgtgctg gcccatcact ttggcaaaga attgggattc gaacatcgat tgaattcgcc
     1321 accatggaga tggcaagcca gcagggcgag acgatgtccg gaccgctgca cattggtttg
     1381 attcccacag ttggaccgta cctgctaccg catattatcc ctatgctgca ccagaccttt
     1441 ccaaagctgg aaatgtatct gcatgaagca cagacccacc agttactggc gcaactggac
     1501 agcggcaaac tcgattgcgt gatcctcgcg ctggtgaaag agagcgaagc attcattgaa
     1561 gtgccgttgt ttgatgagcc aatgttgctg gctatctatg aagatcaccc gtgggcgaac
     1621 cgcgaatgcg taccgatggc cgatctggca ggggaaaaac tgctgatgct ggaagatggt
     1681 cactctttgc gcgatcaggc aatgtccgcc ggctacaaca gcgacaacgt ctatatcatg
     1741 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa cgtcgaggac
     1801 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1861 ctgctgcccg acaaccacta cctgagcttc cagtccgtcc tgagcaaaga ccccaacgag
     1921 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1981 gacgagctgt acaacgtgga tggcggtagc ggtggcaccg gcagcaaggg cgaggagctg
     2041 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2101 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagctgatc
     2161 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct cggctacggc
     2221 ctgaagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2281 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2341 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2401 atcggcttca aggaggacgg caacatcctg gggcacaagc tggagtacaa cggcaccggt
     2461 ttctgttttg aagccggggc ggatgaagat acacacttcc gcgcgaccag cctggaaact
     2521 ctgcgcaaca tggtggcggt aggtagcggg atcactttac tgccagcgct ggctgtgccg
     2581 ccggagcgca aacgcgatgg ggttgtttat ctgccgtgca ttaagccgga accacgccgc
     2641 actattggcc tggtttatcg tcctggctca ccgctgcgca gccgctatga gcagctggca
     2701 gaggccatcc gcgcaagaat ggatggccat ttcgataaag ttttaaaaca ggcggttggt
     2761 ggtagtggtc acagccagaa gagggtggtc gtgctgggct ccggagtcat cgggctctct
     2821 agcgccctga ttctcgctag aaaaggctac tccgtgcata tcctggccag agacttgcct
     2881 gaggatgtca gctcccagac cttcgcaagc ccctgggccg gagctaactg gacaccattt
     2941 atgaccctga cagacggacc cagacaagcc aagtgggaag agtccacctt caagaaatgg
     3001 gtggagctgg ttcccacagg ccacgctatg tggctgaaag ggaccagaag gtttgcccag
     3061 aatgaggatg gcctcctggg acattggtac aaggacatca cccctaacta taggccactt
     3121 ccaagctctg aatgtccacc tggagccatt ggagtgacat acgacaccct gagcgtccac
     3181 gctcccaagt attgccaata ccttgccaga gagctgcaga aacttggggc taccttcgaa
     3241 aggagaacag tgactagcct ggagcaagcc ttcgatggag ctgacctcgt ggtcaacgcc
     3301 accgggctgg gagctaagtc cattgccggc atcgatgacc aggctgccga gcccattagg
     3361 ggacagaccg tgcttgtcaa atctccctgt aagaggtgca caatggatag ctccgaccct
     3421 gcttctcccg cttatatcat tcctagacca ggaggagaag tgatctgtgg cggaacctac
     3481 ggagtcggcg attgggacct gagcgtgaat cccgaaacag tccaaaggat tctcaagcac
     3541 tgcctgagac ttgaccctac cattagctcc gatggaacta tcgagggcat tgaagtgctg
     3601 agacataacg ttggcctgag acccgccagg agaggaggac ctagggtgga ggctgaaaga
     3661 atcgtcttgc ccctggaccg gaccaagagc cctctctctc tgggaagagg gtccgcacgg
     3721 gctgcaaaag agaaggaagt gacccttgtc cacgcctacg gctttagtag cgcagggtat
     3781 cagcaatcct ggggagccgc tgaggacgtg gctcagctgg tcgatgaagc cttccaaaga
     3841 taccacggcg cagctaggga gagcctgccc cccctggagc ggctgaccct gtaagcttgc
     3901 ctcgagcagc gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc
     3961 tctcctggcc ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag
     4021 ttgcatcatt ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt
     4081 atggagcaag gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc
     4141 aagctggagt gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc
     4201 gattctcctg cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag
     4261 ctaatttttg tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa
     4321 ctcctaatct caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg
     4381 aaccactgct cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg
     4441 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     4501 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     4561 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg
     4621 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     4681 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag
     4741 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4801 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4861 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4921 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4981 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     5041 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5101 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5161 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5281 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5341 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5401 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5461 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5521 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5581 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5641 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5701 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5761 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5821 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5881 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5941 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6001 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6061 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt
//
