LOCUS       sequence-268612-        6796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of soma-targeted ChrimsonR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6796)
  AUTHORS   Hage TA, Bosma-Moody A, Baker CA, Kratz MB, Campagnola L, Jarsky T, 
            Zeng H, Murphy GJ
  TITLE     Synaptic connectivity to L2/3 of primary visual cortex measured by 
            two-photon optogenetic stimulation.
  JOURNAL   Elife. 2022 Jan 21;11:e71103. doi: 10.7554/eLife.71103.
  PUBMED    35060903
REFERENCE   2  (bases 1 to 6796)
  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: "2022-01-21"; volume: "11:e71103. doi"; pages: " 
            10.7554/eLife.71103"
COMMENT     Sequence Label: flex-ChrimsonR-EYFP-Kv
FEATURES             Location/Qualifiers
     source          1..6796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      576..1164
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1065..1084
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1226..1355
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        1400..1847
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(1885..1918)
                     /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(1969..2002)
                     /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(2209..2919,2920..2922,2923..2925))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2251..2272)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2600..2619
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2859..2880
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2944..3993)
                     /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"
     regulatory      3990..3999
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    4006..4039
                     /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    4090..4123
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4148..4736
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4201..4221)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4619..4630)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4768..5244
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5284..5424
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5284..5413
                     /label=AAV2 ITR
     rep_origin      5499..5954
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5586..5605)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5796..5817
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5971..5990)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6090..6112
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6150..6168)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6236..6340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6341..6409,6410..6796,1..405)
                     /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(6559..6578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
       61 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
      121 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
      181 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
      241 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
      301 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
      361 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
      421 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
      481 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
      541 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
      601 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
      661 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
      721 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
      781 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
      841 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
      901 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
      961 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1021 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1081 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1141 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1201 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
     1261 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     1321 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtg tgtctagact
     1381 gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg
     1441 gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa
     1501 ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac
     1561 tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc
     1621 gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg
     1681 gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg
     1741 agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc
     1801 agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg agaaggtacc
     1861 ggatcctcta gagtcgactc cggaataact tcgtatagga tactttatac gaagttatgc
     1921 agaatggtag ctggattgta gctgctatta gcaatatgaa acctcttaat aacttcgtat
     1981 agcatacatt atacgaagtt atggcgcgcc ttaaaaccgc gtagcctctg gaaaatcggt
     2041 ggcgcatgag ataaaagagt caatagaact catggacctc atgtcgatca cgccctctgt
     2101 tcttgcagga agaggtgcca caggggaggg cattgaggac atttccagct cctcaggggg
     2161 tttactctgg ggggccatct ctttggtgtt cagaataggc tgggactgct tgtacagctc
     2221 gtccatgccg agagtgatcc cggcggcggt cacgaactcc agcaggacca tgtgatcgcg
     2281 cttctcgttg gggtctttgc tcagggcgga ctggtagctc aggtagtggt tgtcgggcag
     2341 cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag tggtcggcga gctgcacgct
     2401 gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg atgccgttct tctgcttgtc
     2461 ggccatgata tagacgttgt ggctgttgta gttgtactcc agcttgtgcc ccaggatgtt
     2521 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     2581 gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc ttgaagaaga tggtgcgctc
     2641 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     2701 gtagcgggcg aagcactgca ggccgtagcc gaaggtggtc acgagggtgg gccagggcac
     2761 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgtagg tggcatcgcc
     2821 ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     2881 gatgggcacc accccggtga acagctcctc gcccttgctc accattgcta ctaccggtgc
     2941 cgccactgtg tcctcgtcct cctcctccac gaactcttcc acttccacct cctcgcctcc
     3001 gatctccatc ttggtggtct tccggatgtc gccgtggatc aggatgtgct cgtggatctt
     3061 gatcctcagg tggtgggcca ggaaggtcca aaactccttg gcgatgatgt cgcagatgct
     3121 gtggccgatg ctgttggcgt aagggctcag cttcagcagt ccttctggtc ccactgccca
     3181 gaggattggg tagctgcccc aagaggcgaa gtaagcgtag gccatcagct tcacgaccat
     3241 gcggcaatgg cctttaggca cgctgtggtt ggcttccacg tagcacttgg cggcctggaa
     3301 gtacatgtag ccgccgtaga tgcaagacac gatatacagc agccacttga gccaatcggt
     3361 agccagtcct gcggccatgc cgaacacgat cattcccacg caagacacga tcaggcccat
     3421 ggtccgcttg ctgtagtcgt tcttcaggcc gctcaggttg ctcagtctga tcaggatcac
     3481 ggggcaagac agcagccact cgaagtagcg caggcaatag gcgtggttgc cggtagacag
     3541 gtacactgtg gcggggctgc tgaactcctt gaagatctcc agggtcacga acagcacctc
     3601 gacgcaacag acgtagacct cctcccaacc gcaagtggcc ttccaggcgc tgaagccgta
     3661 gaatgtcagc agggcgatgg cgatgctgaa agcaatccac tggcagacct gggcgccgat
     3721 cttttctcct ggtgtgcctc tgctgtgcag ccaagcctgc ttcttctcca ctcccacgac
     3781 caggtagccg cctgtggcct ccacgaagca atatccgtat ccggcgtcgc tcagtccgta
     3841 agaagggtca caccaccact cggtgctgaa gcactcgcct gtaggtgtca ttcctccaca
     3901 gcccatgtcc tcgtggtggt aggggttagg ccaagggttg atgcctccgg cggcaaacag
     3961 agatctggtg gcgctgctga tcagctcagc catggtggcg ctagcataac ttcgtataaa
     4021 gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt ctttagaatg
     4081 gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcg atatcaagct
     4141 tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4201 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4261 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4321 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4381 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4441 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4501 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     4561 gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4621 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4681 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg
     4741 ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc cccagtgcct
     4801 ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt
     4861 tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta
     4921 tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca
     4981 agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg
     5041 attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc
     5101 taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac
     5161 tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga
     5221 accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg accgagcggc
     5281 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     5341 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     5401 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     5461 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     5521 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     5581 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     5641 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5701 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5761 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5821 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     5881 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5941 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     6001 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     6061 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     6121 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     6181 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     6241 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6301 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     6361 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6421 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6481 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6541 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6601 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6661 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6721 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6781 cgcttttttg cacaac
//
