LOCUS       sequence-436454-        7402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent Chr2-mCherry encoded in an AAV vector for rAAV 
            production and expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7402)
  AUTHORS   Tang JC, Drokhlyansky E, Etemad B, Rudolph S, Guo B, Wang S, Ellis 
            EG, Li JZ, Cepko CL
  TITLE     Detection and manipulation of live antigen-expressing cells using 
            conditionally stable nanobodies.
  JOURNAL   Elife. 2016 May 20;5. pii: e15312. doi: 10.7554/eLife.15312.
  PUBMED    27205882
REFERENCE   2  (bases 1 to 7402)
  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: "Elife."; 
            date: "2016-05-20"; volume: "5. pii: e15312. doi"; pages: " 
            10.7554/eLife.15312"
COMMENT     Sequence Label: pAAV-CAG-FLEXFRT-ChR2(H134R)-mCherry
FEATURES             Location/Qualifiers
     source          1..7402
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        223..526
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        528..807
                     /label=chicken beta-actin promoter
     intron          808..1824
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1748..1770
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1832..1851
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1928..1961
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    1972..2005
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     CDS             complement(2022..2729)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2300..2319)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2569..2587
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2742..3668)
                     /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      3665..3674
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3705..3738)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    complement(3749..3782)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     misc_feature    3806..4394
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3859..3879)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4277..4288)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4429..4446)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4435..4642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4665..4805
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4665..4794
                     /label=AAV2 ITR
     rep_origin      4880..5335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4967..4986)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5177..5198
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5352..5371)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5471..5493
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5531..5549)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5617..5721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5722..5790,5791..6582)
                     /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(5940..5959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6753..7341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7242..7261
                     /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 gcgaaacaat tctgcaggaa tctagttatt aatagtaatc
      181 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      241 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      301 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      361 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      421 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      481 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      541 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      601 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggg gcgcgcgcca
      661 ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc
      721 aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc
      781 tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc
      841 cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag
      901 gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg
      961 cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc
     1021 ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc
     1081 tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc
     1141 agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag
     1201 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     1261 tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
     1321 tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
     1381 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
     1441 gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     1501 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag
     1561 ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc
     1621 gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt
     1681 ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc
     1741 agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt
     1801 catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat
     1861 cattttggca aagaattgat ttgataccgc gggccccgcg tggacggtac ccttcaggtc
     1921 gacgtcggaa gttcctattc tctagaaagt ataggaactt cccgaatcgc agaagttcct
     1981 attcttcaaa tagtatagga acttcttggc gcgccgctag cttacttgta cagctcgtcc
     2041 atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat ggtgtagtcc
     2101 tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg cagctgcacg
     2161 ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc gtccttcagc
     2221 ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat ccgctcggag
     2281 gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg gaagttggtg
     2341 ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc ctgggtcacg
     2401 gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc ctcggggaag
     2461 gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt ggagccgtac
     2521 atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt ggtcaccttc
     2581 agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc gatctcgaac
     2641 tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc cttgatgatg
     2701 gccatgttat cctcctcgcc cttgctcacg gcggccgctg gcacggctcc ggcctcggct
     2761 tcgtcttcga cgagagtctc gacctcgatc tccgttccgc cgatgttcag tttggtggtt
     2821 ttgcggatat ctccgtgaat caatatgtgc tcgtggatca ggacgcgcag gtagtgtccc
     2881 aacaaccccc aacaattttt actcatcaga tcaataatcg tgtgacctac ggtggagcca
     2941 tagacgctca ggacgccaaa accttcgggc cccaaaatga agagaattgg gaacataccc
     3001 cagctcacga aaaacagcca tgccatgccg gtcacgacct ggcggcaccg accctttggc
     3061 acagtatgat aaccctcgat atatgctttg gcggcgtgaa aaaatgtgtt cgcgccatag
     3121 cacaatccaa gacaaaagaa gatgacttta acatagccgg ttgccatggc gctggtagcc
     3181 ccccacacga tagtcccgat gtctgagaca aggagtccca tggttctcct gctgtagtcg
     3241 ttgctcaggc cggtgaggtt gctcaggcgg ataaggatga caggacaagt gagcagccac
     3301 tctgcatagc gcagccactg cacccggtgt cctgtggcaa ggtagagcat agagggattc
     3361 ttaaactcaa aaaagaactc gagaatcacc ttaaccattt caatggcgca cacatagatc
     3421 tcctcccagc cgcatgtaga tttccaggtt tggtaggcat agaacatcag cagcaaaatg
     3481 ctgaatcctg ctgcaagcca ctgcaggaca tttgacgcgg tctgagcgcc gttcgtgccg
     3541 cgagattcaa tccatccggc acagtaacat tgatcctcag ggaccaggac ggacccgttc
     3601 accaccacag gattagtaac gaacaaaagt tcgcgtccga cggcagacaa agcgccgcca
     3661 tagtccatgg tggcgctagc ggcgcgccaa cctggccggc cttggaagtt cctatacttt
     3721 ctagagaata ggaacttccc gaatcgcaga agttcctata ctatttgaag aataggaact
     3781 tccgaagatc atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt
     3841 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc
     3901 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg
     3961 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac
     4021 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc
     4081 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc
     4141 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa
     4201 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc
     4261 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc
     4321 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg
     4381 ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tcgatctgcc tcgactgtgc
     4441 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     4501 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     4561 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     4621 acaatagcag gcatgctggg gacacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4681 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4741 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4801 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4861 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4921 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     4981 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5041 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     5101 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5161 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     5221 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     5281 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     5341 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5401 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5461 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5521 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5581 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5641 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5701 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5761 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5821 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5881 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5941 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6001 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6061 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6121 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6181 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6241 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6301 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6361 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6421 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6481 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6541 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6601 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6661 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6721 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6781 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6841 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6901 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6961 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7021 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7081 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7141 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7201 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7261 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7321 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7381 tgctggcctt ttgctcacat gt
//
