LOCUS       sequence-320934-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLiCRE protease with luciferin-gating for neuronal expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Kim CK, Sanchez MI, Hoerbelt P, Fenno LE, Malenka RC, Deisseroth K, 
            Ting AY
  TITLE     A Molecular Calcium Integrator Reveals a Striatal Cell Type Driving 
            Aversion.
  JOURNAL   Cell. 2020 Dec 23;183(7):2003-2019.e16. doi: 
            10.1016/j.cell.2020.11.015. Epub 2020 Dec 11.
  PUBMED    33308478
REFERENCE   2  (bases 1 to 6765)
  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"; doi: 
            "10.1016/j.cell.2020.11.015"; journalName: "Cell"; date: 
            "2020-12-23- 23"; volume: "183"; issue: "7"; pages: "2003-2019.e16"
COMMENT     Sequence Label: GFP-CaM-NanoLuc-15aa-uTEVp
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      255..264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(261..263,264..266,267..977)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(261..263,264..266,267..977)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(306..327)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(567..586)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     914..935
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1014..1457
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             1458..1499
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1530..2039
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     misc_feature    2769..3357
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2822..2842)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3240..3251)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3399..3416)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3405..3612
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3641..3781
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3641..3770
                     /label=AAV2 ITR
     rep_origin      3856..4311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3943..3962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4153..4174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4328..4347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4447..4469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4507..4525)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4593..4697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4698..4766,4767..5558)
                     /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(4916..4935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5729..6317
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6218..6237
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6379..6508
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(6559..6765,1..241)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg tcactcgccg gtcccccgca
       61 aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca
      121 ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga
      181 ctcagcgctg cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca
      241 gtcgagaagg atccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc
      301 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
      361 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
      421 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
      481 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
      541 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
      601 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
      661 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
      721 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
      781 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
      841 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
      901 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
      961 tggacgagct gtacaaggac cttggatctg gcacaggcgg atccggttca ggcatggacc
     1021 aactgactga agagcagatc gcagaattta aagaggcttt ctccctactg gacaaggacg
     1081 gggatgggac aataacaacc aaggagctgg ggacggggat gcggtctctg gggcagaacc
     1141 ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt gacggcacaa
     1201 tcgacttccc tgagttcctg acaatgatgg caagaaaaat gaaatacaca gacagtgaag
     1261 aagaaattag agaagcgttc cgtgtgtttg ataaggatgg caatggctac atcagtgcag
     1321 cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa gaggttgatg
     1381 aaatgatcag ggaagcagac atcgatgggg atggtcaggt aaactacgaa gagtttgtac
     1441 aaatgatgac agcgaagggc aagccgatcc ctaaccctct gctgggcctg gacagcactg
     1501 gtggaagtgg atcaggcagc ggtggatctg tcttcacact cgaagatttc gttggggact
     1561 ggcgacagac agccggctac aacctggacc aagtccttga acagggaggt gtgtccagtt
     1621 tgtttcagaa tctcggggtg tccgtaactc cgatccaaag gattgtcctg agcggtgaaa
     1681 atgggctgaa gatcgacatc catgtcatca tcccgtatga aggtctgagc ggcgaccaaa
     1741 tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt ggatgatcat cactttaagg
     1801 tgatcctgca ctatggcaca ctggtaatcg acggggttac gccgaacatg atcgactatt
     1861 tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa aaagatcact gtaacaggga
     1921 ccctgtggaa cggcaacaaa attatcgacg agcgcctgat caaccccgac ggctccctgc
     1981 tgttccgagt aaccatcaac ggagtgaccg gctggcggct gtgcgaacgc attctggcgg
     2041 gcgacccact agtccagtgt ggtggaattg ccggatccgc caccggagaa tccttgttta
     2101 agggaccacg tgattacaac ccgatatcga gcaccatttg tcatttgacg aatgaatctg
     2161 atgggcacac aacatcgttg tatggtattg gatttggtcc cttcatcatt acaaacaagc
     2221 acttgtttag aagaaataat ggaacactgt tggtccaatc actacatggt gtattcaagg
     2281 tcaagaacac cacgactttg caacaacacc tcattgatgg gagggacatg ataattattc
     2341 gcatgcctaa ggatttccca ccatttcctc aaaagctgaa atttagagag ccacaaaggg
     2401 aagagcgcat atgtcttgtg acaaccaact tccaaactaa gagcatgtct agcatggtgt
     2461 cagacactag ttgcacattc ccttcatctg atggcatatt ctggaagcat tggattcaaa
     2521 ccaaggatgg gcagtgtggc aatccattag tatcaactag agatgggttc attgttggta
     2581 tacactcagc atcgaatttc accaacacaa acaattattt cacaagcgtg ccgaaaaact
     2641 tcatggaatt gttgacaaat caggaggcgc agcagtgggt tagtggttgg cgattaaatg
     2701 ctgactcagt attgtggggg ggccataaag ttttcatggt gtaagaattc gatatcaagc
     2761 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     2821 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     2881 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     2941 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3001 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3061 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3121 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3181 ggctgctcgc ctatgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3241 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3301 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3361 gataccgagc gctgctcgag ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     3421 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3481 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3541 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3601 gcatgctggg gaggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     3661 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3721 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3781 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3841 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3901 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     3961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4201 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4321 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4381 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4441 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4501 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4561 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5401 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5641 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5701 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5761 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5821 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5881 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5941 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6001 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6061 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6121 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6181 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6241 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6301 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6361 ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc
     6421 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
     6481 gtggccaact ccatcactag gggttcctgc ggccgcacgc gttgtacagt gtctagactg
     6541 cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc ggggtggggg
     6601 tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat
     6661 tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact
     6721 gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcgg
//
