LOCUS       sequence-230184-        7366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRuby2 in the nucleus of Cre-negative cells, and 
            ChR(H134R)-YFP in Cre-positive cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7366)
  AUTHORS   Wozny C, Beed P, Nitzan N, Possnecker Y, Rost BR, Schmitz D
  TITLE     VGLUT2 Functions as a Differential Marker for Hippocampal Output 
            Neurons.
  JOURNAL   Front Cell Neurosci. 2018 Oct 2;12:337. doi: 
            10.3389/fncel.2018.00337. eCollection 2018.
  PUBMED    30333731
REFERENCE   2  (bases 1 to 7366)
  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: "Front Cell 
            Neurosci."; date: "2018-10-2"; volume: "12:337. doi"; pages: " 
            10.3389/fncel.2018.00337. eCollection 2018"
COMMENT     Sequence Label: 
            pAAV-EF1a-SwitchON_mRubyNLS-hChR2(H134R)-EYFP-NO_WPRE
FEATURES             Location/Qualifiers
     source          1..7366
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1434..1467)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     regulatory      1474..1483
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1486..1506
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1513..2220
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     protein_bind    complement(2229..2262)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(2274..2987,2988..2990,2991..2993))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2319..2340)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2668..2687
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2927..2948
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2990..2999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3009..3935)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      3932..3941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3946..3979
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4030..4063
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    4113..4589
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4312..4331)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4629..4769
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4629..4758
                     /label=AAV2 ITR
     rep_origin      4844..5299
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4931..4950)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5141..5162
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5316..5335)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5435..5457
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5495..5513)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5581..5685
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5686..5754,5755..6546)
                     /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(5904..5923)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6717..7305
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7206..7225
                     /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 gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gacataactt
     1441 cgtataggat actttatacg aagttatcct agggccacca tggccccaaa gaagaagcgg
     1501 aaggtcacta gtgtgtctaa gggcgaagag ctgatcaagg aaaatatgcg tatgaaggtg
     1561 gtcatggaag gttcggtcaa cggccaccaa ttcaaatgca caggtgaagg agaaggcaat
     1621 ccgtacatgg gaactcaaac catgaggatc aaagtcatcg agggaggacc cctgccattt
     1681 gcctttgaca ttcttgccac gtcgttcatg tatggcagcc gtacttttat caagtacccg
     1741 aaaggcattc ctgatttctt taaacagtcc tttcctgagg gttttacttg ggaaagagtt
     1801 acgagatacg aagatggtgg agtcgtcacc gtcatgcagg acaccagcct tgaggatggc
     1861 tgtctcgttt accacgtcca agtcagaggg gtaaactttc cctccaatgg tcccgtgatg
     1921 cagaagaaga ccaagggttg ggagcctaat acagagatga tgtatccagc agatggtggt
     1981 ctgaggggat acactcatat ggcactgaaa gttgatggtg gtggccatct gtcttgctct
     2041 ttcgtaacaa cttacaggtc aaaaaagacc gtcgggaaca tcaagatgcc cggtatccat
     2101 gccgttgatc accgcctgga aaggttagag gaaagtgaca atgaaatgtt cgtagtacaa
     2161 cgcgaacacg cagttgccaa gttcgccggg cttggtggtg ggatggacga gctgtacaag
     2221 gattatgaat aacttcgtat agcatacatt atacgaagtt atggcgcgcc ctattacttg
     2281 tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg
     2341 tgatcgcgct tctcgttggg gtctttgctc agggcggact ggtagctcag gtagtggttg
     2401 tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc
     2461 tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat gccgttcttc
     2521 tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc
     2581 aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg
     2641 tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg
     2701 gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg
     2761 tggtcggggt agcgggcgaa gcactgcagg ccgtagccga aggtggtcac gagggtgggc
     2821 cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg
     2881 gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc gccgtccagc
     2941 tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac catggtggcg
     3001 gccgctggca cggctccggc ctcggcttcg tcttcgacga gagtctcgac ctcgatctcc
     3061 gttccgccga tgttcagttt ggtggttttg cggatatctc cgtgaatcaa tatgtgctcg
     3121 tggatcagga cgcgcaggta gtgtcccaac aacccccaac aatttttact catcagatca
     3181 ataatcgtgt gacctacggt ggagccatag acgctcagga cgccaaaacc ttcgggcccc
     3241 aaaatgaaga gaattgggaa cataccccag ctcacgaaaa acagccatgc catgccggtc
     3301 acgacctggc ggcaccgacc ctttggcaca gtatgataac cctcgatata tgctttggcg
     3361 gcgtgaaaaa atgtgttcgc gccatagcac aatccaagac aaaagaagat gactttaaca
     3421 tagccggttg ccatggcgct ggtagccccc cacacgatag tcccgatgtc tgagacaagg
     3481 agtcccatgg ttctcctgct gtagtcgttg ctcaggccgg tgaggttgct caggcggata
     3541 aggatgacag gacaagtgag cagccactct gcatagcgca gccactgcac ccggtgtcct
     3601 gtggcaaggt agagcataga gggattctta aactcaaaaa agaactcgag aatcacctta
     3661 accatttcaa tggcgcacac atagatctcc tcccagccgc atgtagattt ccaggtttgg
     3721 taggcataga acatcagcag caaaatgctg aatcctgctg caagccactg caggacattt
     3781 gacgcggtct gagcgccgtt cgtgccgcga gattcaatcc atccggcaca gtaacattga
     3841 tcctcaggga ccaggacgga cccgttcacc accacaggat tagtaacgaa caaaagttcg
     3901 cgtccgacgg cagacaaagc gccgccatag tccatggtgg ctagcataac ttcgtataaa
     3961 gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg
     4021 gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct
     4081 tatcgatacc gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag
     4141 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     4201 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     4261 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     4321 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     4381 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     4441 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     4501 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     4561 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     4621 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4681 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4741 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4801 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4861 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4921 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4981 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     5041 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     5101 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     5161 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     5221 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     5281 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     5341 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     5401 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5461 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5521 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5581 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5641 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5701 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5761 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5821 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5881 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5941 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     6001 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     6061 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     6121 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     6181 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     6241 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     6301 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     6361 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6421 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6481 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6541 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6601 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6661 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6721 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6781 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6841 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6901 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6961 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7021 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7081 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7141 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7201 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     7261 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     7321 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
