LOCUS       sequence-272778-        7098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Intersectional viral expression of ChRmine-p2a-oScarlet in cells 
            expressing Flp AND NOT Cre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7098)
  AUTHORS   Fenno LE, Ramakrishnan C, Kim YS, Evans KE, Lo M, Vesuna S, Inoue M,
            Cheung KYM, Yuen E, Pichamoorthy N, Hong ASO, Deisseroth K
  TITLE     Comprehensive Dual- and Triple-Feature Intersectional Single-Vector 
            Delivery of Diverse Functional Payloads to Cells of Behaving 
            Mammals.
  JOURNAL   Neuron. 2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003.
  PUBMED    32574559
REFERENCE   2  (bases 1 to 7098)
  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: "Neuron. 
            2020 Jun 20. pii: S0896-6273(20)30432-3. doi: 
            10.1016/j.neuron.2020.06.003."
COMMENT     Sequence Label: pAAV-nEF-Coff/Fon-ChRmine-oScarlet
FEATURES             Location/Qualifiers
     source          1..7098
                     /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        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     protein_bind    697..730
                     /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)"
     primer_bind     complement(1125..1144)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(1408..1441)
                     /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    complement(1492..1525)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     primer_bind     1606..1624
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(1758..1814)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    2597..2630
                     /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    2681..2714
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     regulatory      3067..3076
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3083..3116)
                     /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    3225..3813
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3278..3298)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3696..3707)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3845..4321
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4044..4063)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4361..4501
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4361..4490
                     /label=AAV2 ITR
     rep_origin      4576..5031
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4663..4682)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4873..4894
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5048..5067)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5167..5189
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5227..5245)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5313..5417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5418..5486,5487..6278)
                     /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(5636..5655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6449..7037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6938..6957
                     /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 gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgaag ttcctattct tcaaatagta
      721 taggaacttc gcagagtagt agctggattg tagctgctat tagcaatcta aaacctctta
      781 gaagttccta ttcttcaaaa ggtataggaa cttcggcgcg cctcattaca cctcgttctc
      841 gtagcagaac ttgtacagct cgtccatgcc gccggtggag tggcggccct cggagcgttc
      901 gtactgttcc accacggtgt agtcctcgtt gtgggaggtg atgtccaact tgcggtccac
      961 gttgtaggcg ccgggcatct gcacgggctt cttggccttg taggtggtct tgaagtccgc
     1021 caggtagcgc ccgccgtcct tcaggcgcag ggccatctta atgtcgccct tcagcacgcc
     1081 gtcctcgggg tacaaccgct cggtggacgc ttcccagccc attgtcttct tctgcattac
     1141 ggggccgtca ggagggaagt tggtgccgcg gagcttcacc ttgtagatca gggtgccgtc
     1201 ctccagggag gtgtcctggg tcacggtcac ggcgccgccg tcctcgaagt tcatcacgcg
     1261 gtcccacttg aagccctcgg ggaaggactg cttatagtag tcggggatgt cggcggggtg
     1321 cttggtgaag gccctgtggg acgacatgac ttaaccagtt aaccatgttc tctgaacatg
     1381 agaagggctg gccactctcc agtcgacata acttcgtata ggatacttta tacgaagtta
     1441 tgcagaatgg tagctggatt gtagctgcta ttagcaatat gaaacctctt aataacttcg
     1501 tatagtatac cttatacgaa gttatactag tcctcctgta ctcacctgga gccgtacatg
     1561 aactgagggg acaggatgtc ccaggagaag ggcagggggc cacccttggt caccttcagc
     1621 ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg
     1681 tggccgttca tggagccctc catgtgcacc ttgaaccgca tgaactcctt gatcactgcc
     1741 tcgcccttgc tcaccatggg accggggttt tcttccacgt ctcctgcttg ctttaacaga
     1801 gagaagttcg tggctccgga gccgggcgcg cccacgttga tgtcgatctg gtccaggggg
     1861 atgtactcgc cctcgctggt gatcctgctc ttggcggccg cagacagtct ccgcaggcgc
     1921 tcgccattgc tgctcagcct catatccagc tccagcttct ggaactcatc ctcgtcgtgt
     1981 cccagctgct ccagcagccg aaacagctcc tcatcatagg tctttctgaa ccgggccagg
     2041 gcaaagccgt aacaggactt ggccacgatg tcgcacacgc agtgggccac ctctgtccag
     2101 ttatcgtcga tccagccgaa gcctctaggg cagatggccc acaccagggg aaacagaatc
     2161 cacagcatgg agaaggtaaa cacggcgaac ttcagcacgt gcaggctgtg ctcggcgcct
     2221 gtatcctggg ccagctgggc cagccggcta tactgcttgg ccacgatgct catcacgata
     2281 gagtaggcga agataaacca gaaacagcca aagccatacc aggcgtaggc gccattcctc
     2341 agcctagggt cgccctctgc gtagaactcg gccagcacgc cagacatcag gatggcaaag
     2401 atgatggcgc tacaagacac gcgatagggg gccctcagct gatacagcag atcgtacacc
     2461 agcattgggc aggtcagcat gtagtcggca tacctaaaca catgagaaat agagtcataa
     2521 gcacatgggt cacatacagg agatatgtat ataacattaa tacaatttta taaaaaaaag
     2581 ggggggcaca ggtaccataa cttcgtataa agtatcctat acgaagttat ttgccttaac
     2641 ccagaaatta tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataaggtata
     2701 ctatacgaag ttatgctagc aaccccgaca cttacctgta attgccgaag ccgatgatga
     2761 aggggcccac atcgccgaag ttgtggccca gtgtgtcggc cacgatcttg ccgaagtagc
     2821 tcagcatggc aaagaaattc acaaacaggg ccaggttctg ctcctcctga tagccggcgt
     2881 aaaaggaggt tctcttgcta gggccccgag atgtccagga ctcatagccc aggtagaatg
     2941 tggtgattgt cagcacgata aagcaagacc agtgggcgcc gatggccacg gagttcagct
     3001 tggtatccag ataccagccg tccattgtgc ccacgtagaa catctggtcg gtgcctggtg
     3061 cgtgtgccat ggtggcgagc tcgaagttcc tatactattt gaagaatagg aacttcttgc
     3121 cttaacccag aaattatcac tgttattctt tagaatggtg caaagagaag ttcctatacc
     3181 ttttgaagaa taggaacttc gaattcgata tcaagcttat cgataatcaa cctctggatt
     3241 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     3301 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     3361 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     3421 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     3481 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     3541 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3601 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct
     3661 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3721 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3781 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagagat
     3841 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     3901 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     3961 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     4021 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     4081 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     4141 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     4201 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     4261 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     4321 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     4381 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     4441 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     4501 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     4561 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4621 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     4681 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4741 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4801 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4861 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4921 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4981 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     5041 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     5101 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     5161 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     5221 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     5281 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5341 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5401 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5461 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5521 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5581 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5641 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5701 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5761 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5821 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5881 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5941 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6001 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6061 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6121 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6181 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6241 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6301 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6361 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6421 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6481 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6541 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6601 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6661 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6721 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6781 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6841 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6901 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6961 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7021 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7081 ggccttttgc tcacatgt
//
