LOCUS       sequence-181305-        5481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protease component of FLARE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5481)
  AUTHORS   Wang W, Wildes CP, Pattarabanjird T, Sanchez MI, Glober GF, Matthews
            GA, Tye KM, Ting AY
  TITLE     A light- and calcium-gated transcription factor for imaging and 
            manipulating activated neurons.
  JOURNAL   Nat Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3909.
  PUBMED    28650461
REFERENCE   2  (bases 1 to 5481)
  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: "Nat 
            Biotechnol. 2017 Jun 26. doi: 10.1038/nbt.3909."
COMMENT     Sequence Label: Ca-FLARE (protease)
FEATURES             Location/Qualifiers
     source          1..5481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      254..263
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             260..703
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             704..745
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1484..2072
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1537..1557)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1955..1966)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2114..2131)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2120..2327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2356..2496
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2356..2485
                     /label=AAV2 ITR
     rep_origin      2571..3026
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2658..2677)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2868..2889
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3043..3062)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3162..3184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3222..3240)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3308..3412
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3413..3481,3482..4273)
                     /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(3631..3650)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4444..5032
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4933..4952
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5094..5223
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(5274..5481,1..240)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa
       61 actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac
      121 cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac
      181 tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag
      241 tcgagaagga tccgccacca tggaccaact gactgaagag cagatcgcag aatttaaaga
      301 ggctttctcc ctactggaca aggacgggga tgggacaata acaaccaagg agctggggac
      361 ggggatgcgg tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga
      421 agtagatgcc gacggtgacg gcacaatcga cttccctgag ttcctgacaa tgatggcaag
      481 aaaaatgaaa tacacagaca gtgaagaaga aattagagaa gcgttccgtg tgtttgataa
      541 ggatggcaat ggctacatca gtgcagcaga gcttcgccac gtgatgacaa accttggaga
      601 gaagttaaca gatgaagagg ttgatgaaat gatcagggaa gcagacatcg atggggatgg
      661 tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg aagggcaagc cgatccctaa
      721 ccctctgctg ggcctggaca gcactggtgg aagtggatca ggcagcggtg gatcttacgg
      781 aagccatgtg gattacgccg gagaaagctt gtttaaggga ccacgtgatt acaacccgat
      841 atcgagcacc atttgtcatt tgacgaatga atctgatggg cacacaacat cgttgtatgg
      901 tattggattt ggtcccttca tcattacaaa caagcacttg tttagaagaa ataatggaac
      961 actgttggtc caatcactac atggtgtatt caaggtcaag aacaccacga ctttgcaaca
     1021 acacctcatt gatgggaggg acatgataat tattcgcatg cctaaggatt tcccaccatt
     1081 tcctcaaaag ctgaaattta gagagccaca aagggaagag cgcatatgtc ttgtgacaac
     1141 caacttccaa actaagagca tgtctagcat ggtgtcagac actagttgca cattcccttc
     1201 atctgatggc atattctgga agcattggat tcaaaccaag gatgggcagt gtggcagtcc
     1261 attagtatca actagagatg ggttcattgt tggtatacac tcagcatcga atttcaccaa
     1321 cacaaacaat tatttcacaa gcgtgccgaa aaacttcatg gaattgttga caaatcagga
     1381 ggcgcagcag tgggttagtg gttggcgatt aaatgctgac tcagtattgt gggggggcca
     1441 taaagttttc atggtgtaag aattcgatat caagcttatc gataatcaac ctctggatta
     1501 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     1561 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     1621 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     1681 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     1741 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     1801 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     1861 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     1921 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     1981 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     2041 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagctaga
     2101 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2161 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     2221 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     2281 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggaggt aaccacgtgc
     2341 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     2401 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     2461 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     2521 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     2581 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2641 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2701 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2761 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     2821 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     2881 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     2941 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3001 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     3061 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3121 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3181 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     3241 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     3301 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3361 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     3421 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     3481 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     3541 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     3601 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     3661 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     3721 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     3781 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     3841 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     3901 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     3961 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     4021 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     4081 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     4141 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     4201 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     4261 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     4321 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     4381 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4441 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4501 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4561 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4621 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4681 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4741 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4801 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4861 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4921 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4981 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5041 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     5101 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
     5161 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     5221 cctgcggccg cacgcgttgt acagtgtcta gactgcagag ggccctgcgt atgagtgcaa
     5281 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac
     5341 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg
     5401 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg
     5461 ccttcgcccc cgcctggcgg c
//
