LOCUS       sequence-267518-        6234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuronal expression of uFLARE protease component.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6234)
  AUTHORS   Sanchez MI, Ting AY
  TITLE     Directed evolution improves the catalytic efficiency of TEV 
            protease.
  JOURNAL   Nat Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7.
  PUBMED    31819267
REFERENCE   2  (bases 1 to 6234)
  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 
            Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7."
COMMENT     Sequence Label: Syn:eGFP-CaM-uTEV1-Delta(220-242)
FEATURES             Location/Qualifiers
     source          1..6234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..498
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      512..521
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(518..520,521..523,524..1234)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(518..520,521..523,524..1234)
                     /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(563..584)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(824..843)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1171..1192
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1271..1714
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             1715..1756
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    2495..3083
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2548..2568)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2966..2977)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3125..3142)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3131..3338
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3367..3507
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3367..3496
                     /label=AAV2 ITR
     rep_origin      3582..4037
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3669..3688)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3879..3900
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4054..4073)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4173..4195
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4233..4251)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4319..4423
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4424..4492,4493..5284)
                     /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(4642..4661)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5455..6043
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5944..5963
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6105..6234
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gcggccgcac gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg agtgcaagtg
       61 ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca
      121 ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg
      181 ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct
      241 tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca
      301 ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc
      361 ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg
      421 cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg
      481 tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg gtgagcaagg gcgaggagct
      541 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
      601 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
      661 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
      721 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
      781 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
      841 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
      901 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
      961 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     1021 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     1081 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     1141 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1201 cgggatcact ctcggcatgg acgagctgta caaggacctt ggatctggca caggcggatc
     1261 cggttcaggc atggaccaac tgactgaaga gcagatcgca gaatttaaag aggctttctc
     1321 cctactggac aaggacgggg atgggacaat aacaaccaag gagctgggga cggggatgcg
     1381 gtctctgggg cagaacccca cagaagcaga gctgcaggac atgatcaatg aagtagatgc
     1441 cgacggtgac ggcacaatcg acttccctga gttcctgaca atgatggcaa gaaaaatgaa
     1501 atacacagac agtgaagaag aaattagaga agcgttccgt gtgtttgata aggatggcaa
     1561 tggctacatc agtgcagcag agcttcgcca cgtgatgaca aaccttggag agaagttaac
     1621 agatgaagag gttgatgaaa tgatcaggga agcagacatc gatggggatg gtcaggtaaa
     1681 ctacgaagag tttgtacaaa tgatgacagc gaagggcaag ccgatcccta accctctgct
     1741 gggcctggac agcactggtg gaagtggatc aggcagcggt ggatcttacg gaagccatgt
     1801 ggattacgcc ggagaatcct tgtttaaggg accacgtgat tacaacccga tatcgagcac
     1861 catttgtcat ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg gtattggatt
     1921 tggtcccttc atcattacaa acaagcactt gtttagaaga aataatggaa cactgttggt
     1981 ccaatcacta catggtgtat tcaaggtcaa gaacaccacg actttgcaac aacacctcat
     2041 tgatgggagg gacatgataa ttattcgcat gcctaaggat ttcccaccat ttcctcaaaa
     2101 gctgaaattt agagagccac aaagggaaga gcgcatatgt cttgtgacaa ccaacttcca
     2161 aactaagagc atgtctagca tggtgtcaga cactagttgc acattccctt catctgatgg
     2221 catattctgg aagcattgga ttcaaaccaa ggatgggcag tgtggcaatc cattagtatc
     2281 aactagagat gggttcattg ttggtataca ctcagcatcg aatttcacca acacaaacaa
     2341 ttatttcaca agcgtgccga aaaacttcat ggaattgttg acaaatcagg aggcgcagca
     2401 gtgggttagt ggttggcgat taaatgctga ctcagtattg tgggggggcc ataaagtttt
     2461 catggtgtaa gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg
     2521 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc
     2581 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta
     2641 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt
     2701 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca
     2761 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc
     2821 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt
     2881 gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg
     2941 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg
     3001 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat
     3061 ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagctag agctcgctga
     3121 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     3181 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     3241 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     3301 ggggaggatt gggaagacaa tagcaggcat gctggggagg taaccacgtg cggaccgagc
     3361 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     3421 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     3481 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     3541 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     3601 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     3661 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3721 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3781 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3841 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3901 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     3961 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4021 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4081 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4141 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4201 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     4261 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4321 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4381 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     4441 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     4501 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     4561 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     4621 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     4681 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     4741 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     4801 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     4861 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     4921 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     4981 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     5041 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     5101 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     5161 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     5221 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     5281 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     5341 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5401 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5461 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5521 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5581 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5641 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5701 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5761 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5821 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5881 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5941 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6001 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6061 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
     6121 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     6181 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcct
//
