LOCUS       sequence-414075-        7399 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cre- and Flp-dependent transgene expression (CRE-ON / FLP-ON) of 
            mRuby-tagged Chrimson in neurons under the control of the hSyn1 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7399)
  AUTHORS   Cardin Lab
  TITLE     Cre- and Flp-dependent transgene expression (CRE-ON / FLP-ON) of 
            mRuby-tagged Chrimson in neurons under the control of the hSyn1 
            promoter
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7399)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-VTKS4-Chrimson-mRuby
FEATURES             Location/Qualifiers
     source          1..7399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   8..137
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        165..612
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    636..669
                     /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)"
     polyA_signal    715..763
                     /note="synthetic polyadenylation signal"
     polyA_signal    772..1248
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(971..990)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(1283..1302)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1327..1375
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    1535..1568
                     /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(1575..1608)
                     /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(1659..1692)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1920..2630)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             complement(2643..3692)
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     protein_bind    3721..3754
                     /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    3805..3838
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3847..4435
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3900..3920)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4318..4329)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4443..4667
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4669..4809
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4669..4798
                     /label=AAV2 ITR
     rep_origin      4884..5339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4971..4990)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5181..5202
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5356..5375)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5475..5497
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5535..5553)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5621..5725
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5726..5794,5795..6586)
                     /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(5944..5963)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6757..7345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7246..7265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
       61 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      121 catcactagg ggttcctgct agactgcaga gggccctgcg tatgagtgca agtgggtttt
      181 aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga cccactggac
      241 aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg gaggggaaac
      301 aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt gccttcgccc
      361 ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc
      421 ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc cgccggccca
      481 gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc
      541 ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc
      601 ctgagagcgc agtcagatat cgtttaaact ccggagaagt tcctattctc tagaaagtat
      661 aggaacttcc ctcagcacca tggctagcgg cagcctcgga gtttgaatag atagaataaa
      721 atatctttat tttcattcca tctgtgtgtt ggttttttgt gtgagatcta cgggtggcat
      781 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
      841 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
      901 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
      961 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     1021 atctccgcct cctgggttca agcgattctc ctgcctcagg ctcccgagtt gttgggattc
     1081 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     1141 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     1201 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gcctcagcta
     1261 attgagtagg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     1321 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     1381 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     1441 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     1501 gggctcgcag ccaacgtcgg ggcggcaggc cgtggaagtt cctattctct agaaagtata
     1561 ggaacttcac cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag
     1621 ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt
     1681 atacgaagtt ataacgcgcc actagtgaat tcttagaatc tggtggcttc agggaagtcc
     1741 gtggcacagc tgatgaagct gtcaatgctg gacatgctcc gcatgtcgat gacgccctcc
     1801 gtgcgtgcgg gcagaggggc cacggggctg ggcatgctgc tcatctccag ctcctctgga
     1861 ggcttgctct gcggggccat ctccttggtg ttgaggatgg gctgggactg ctctagggcc
     1921 ttgtacagct cgtccatccc accaccaagc ccggcgaact tggcaactgc gtgttcgcgt
     1981 tgtactacga acatttcatt gtcactttcc tctaaccttt ccaggcggtg atcaacggca
     2041 tggataccgg gcatcttgat gttcccgacg gtcttttttg acctgtaagt tgttacgaaa
     2101 gagcaagaca gatggccacc accatcaact ttcagtgcca tatgagtgta tcccctcaga
     2161 ccaccatctg ctggatacat catctctgta ttaggctccc aacccttggt cttcttctgc
     2221 atcacgggac cattggaggg aaagtttacc cctctgactt ggacgtggta aacgagacag
     2281 ccatcctcaa ggctggtgtc ctgcatgacg gtgacgactc caccatcttc gtatctcgta
     2341 actctttccc aagtaaaacc ctcaggaaag gactgtttaa agaaatcagg aatgcctttc
     2401 gggtacttga taaaagtacg gctgccatac atgaacgacg tggcaagaat gtcaaaggca
     2461 aatggcaggg gtcctccctc gatgactttg atcctcatgg tttgagttcc catgtacgga
     2521 ttgccttctc cttcacctgt gcatttgaat tggtggccgt tgaccgaacc ttccatgacc
     2581 accttcatac gcatattttc cttgatcagc tcttcgccct tagacaccat taccggtgcc
     2641 gccactgtgt cctcgtcctc ctcctccacg aactcttcca cttccacctc ctcgcctccg
     2701 atctccatct tggtggtctt ccggatgtcg ccgtggatca ggatgtgctc gtggatcttg
     2761 atcctcaggt ggtgggccag gaaggtccaa aactccttgg cgatgatgtc gcagatgctg
     2821 tggccgatgc tgttggcgta agggctcagc ttcagcagtc cttctggtcc cactgcccag
     2881 aggattgggt agctgcccca agaggcgaag taagcgtagg ccatcagctt cacgaccatg
     2941 cggcaatggc ctttaggcac gctgtggttg gcttccacgt agcacttggc ggcctggaag
     3001 tacatgtagc cgccgtagat gcaagacacg atatacagca gccacttgag ccaatcggta
     3061 gccagtcctg cggccatgcc gaacacgatc attcccacgc aagacacgat caggcccatg
     3121 gtccgcttgc tgtagtcgtt cttcaggccg ctcaggttgc tcagtctgat caggatcacg
     3181 gggcaagaca gcagccactc gaagtagcgc aggcaatagg cgtggttgcc ggtagacagg
     3241 tacactgtgg cggggctgct gaactccttg aagatctcca gggtcacgaa cagcacctcg
     3301 acgcaacaga cgtagacctc ctcccaaccg caagtggcct tccaggcgct gaagccgtag
     3361 aatgtcagca gggcgatggc gatgctgaaa gcaatccact ggcagacctg ggcgccgatc
     3421 ttttctcctg gtgtgcctct gctgtgcagc caagcctgct tcttctccac tcccacgacc
     3481 aggtagccgc ctgtggcctc cacgaagcaa tatccgtatc cggcgtcgct cagtccgtaa
     3541 gaagggtcac accaccactc ggtgctgaag cactcgcctg taggtgtcat tcctccacag
     3601 cccatgtcct cgtggtggta ggggttaggc caagggttga tgcctccggc ggcaaacaga
     3661 gatctggtgg cgctgctgat cagctcagcc atggtaagct ttactagtgg tggcgctagc
     3721 ataacttcgt ataaagtatc ctatacgaag ttatttgcct taacccagaa attatcactg
     3781 ttattcttta gaatggtgca aagaataact tcgtataatg tatgctatac gaagttatgg
     3841 cgcgccaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3901 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3961 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     4021 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     4081 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     4141 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     4201 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg
     4261 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     4321 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     4381 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga
     4441 tcctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4501 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4561 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4621 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggcag gaacccctag
     4681 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     4741 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     4801 gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4861 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4921 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc
     4981 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     5041 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt
     5101 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5161 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat
     5221 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa
     5281 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt
     5341 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc
     5401 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5461 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5521 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     5581 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     5641 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     5701 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     5761 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     5821 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     5881 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     5941 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6001 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6061 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6121 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6181 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6241 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6301 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6361 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6421 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6481 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6541 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6601 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     6661 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     6721 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6781 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6841 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6901 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     6961 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7021 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7081 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7141 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7201 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7261 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7321 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7381 cttttgctgg ccttttgct
//
