LOCUS       sequence-261624-        6233 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 6233)
  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 6233)
  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..6233
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      642..651
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(648..650,651..653,654..1364)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(648..650,651..653,654..1364)
                     /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(693..714)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(954..973)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1301..1322
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1401..1844
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             1845..1886
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    2625..3213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2678..2698)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3096..3107)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3255..3272)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3261..3468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3497..3637
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3497..3626
                     /label=AAV2 ITR
     rep_origin      3711..4166
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3798..3817)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4008..4029
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4183..4202)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4302..4324
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4362..4380)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4448..4552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4553..4621,4622..5413)
                     /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(4771..4790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5584..6172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6073..6092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cgccaccatg gtgagcaagg
      661 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
      721 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
      781 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
      841 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
      901 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
      961 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1021 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1081 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1141 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1201 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1261 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1321 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caaggacctt ggatctggca
     1381 caggcggatc cggttcaggc atggaccaac tgactgaaga gcagatcgca gaatttaaag
     1441 aggctttctc cctactggac aaggacgggg atgggacaat aacaaccaag gagctgggga
     1501 cggggatgcg gtctctgggg cagaacccca cagaagcaga gctgcaggac atgatcaatg
     1561 aagtagatgc cgacggtgac ggcacaatcg acttccctga gttcctgaca atgatggcaa
     1621 gaaaaatgaa atacacagac agtgaagaag aaattagaga agcgttccgt gtgtttgata
     1681 aggatggcaa tggctacatc agtgcagcag agcttcgcca cgtgatgaca aaccttggag
     1741 agaagttaac agatgaagag gttgatgaaa tgatcaggga agcagacatc gatggggatg
     1801 gtcaggtaaa ctacgaagag tttgtacaaa tgatgacagc gaagggcaag ccgatcccta
     1861 accctctgct gggcctggac agcactggtg gaagtggatc aggcagcggt ggatcttacg
     1921 gaagccatgt ggattacgcc ggagaatcct tgtttaaggg accacgtgat tacaacccga
     1981 tatcgagcac catttgtcat ttgacgaatg aatctgatgg gcacacaaca tcgttgtatg
     2041 gtattggatt tggtcccttc atcattacaa acaagcactt gtttagaaga aataatggaa
     2101 cactgttggt ccaatcacta catggtgtat tcaaggtcaa gaacaccacg actttgcaac
     2161 aacacctcat tgatgggagg gacatgataa ttattcgcat gcctaaggat ttcccaccat
     2221 ttcctcaaaa gctgaaattt agagagccac aaagggaaga gcgcatatgt cttgtgacaa
     2281 ccaacttcca aactaagagc atgtctagca tggtgtcaga cactagttgc acattccctt
     2341 catctgatgg catattctgg aagcattgga ttcaaaccaa ggatgggcag tgtggcaatc
     2401 cattagtatc aactagagat gggttcattg ttggtataca ctcagcatcg aatttcacca
     2461 acacaaacaa ttatttcaca agcgtgccga aaaacttcat ggaattgttg acaaatcagg
     2521 aggcgcagca gtgggttagt ggttggcgat taaatgctga ctcagtattg tgggggggcc
     2581 ataaagtttt catggtgtaa gaattcgata tcaagcttat cgataatcaa cctctggatt
     2641 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     2701 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     2761 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     2821 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     2881 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     2941 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     3001 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct
     3061 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     3121 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     3181 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg ctcgagctag
     3241 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3301 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3361 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3421 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggagg taaccacgtg
     3481 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3541 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3601 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3661 cttacgcatc tgtgcggtat tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     3721 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3781 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3841 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3901 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     3961 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4021 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     4081 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4141 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     4201 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4261 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4321 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     4381 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     4441 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4501 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4561 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4621 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4681 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4741 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4801 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4861 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4921 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4981 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5041 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5101 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5161 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5221 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     5281 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5341 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5401 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5461 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5521 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5701 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6181 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
