LOCUS       sequence-261625-        6363 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Neuronal expression of uFLARE TF component.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6363)
  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 6363)
  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:NRX-TM-Nav1.6-CIBN-2xMKII-hLOV-TEcs(ENLYFQ/M)-tTA
FEATURES             Location/Qualifiers
     source          1..6363
                     /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)"
     CDS             811..837
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1063..1572
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCN
                     EAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     CDS             2287..2307
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2320..2343
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2344..2964,2965..3351)
                     /codon_start=1
                     /product="tetracycline-controlled transactivator, 
                     comprising a fusion of the tetracycline repressor TetR with
                     the C-terminal activation domain of herpes simplex virus 
                     VP16"
                     /label=tTA
                     /note="In the Tet-Off(R) system, tTA binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTI
                     EGLLDLPDDDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHA
                     DALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     primer_bind     complement(2417..2436)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3385..3402)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3391..3598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3627..3767
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3627..3756
                     /label=AAV2 ITR
     rep_origin      3841..4296
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3928..3947)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4138..4159
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4313..4332)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4432..4454
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4492..4510)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4578..4682
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4683..4751,4752..5543)
                     /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(4901..4920)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5714..6302
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6203..6222
                     /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 gagaagaagc ttgccaccat gcacctgaga
      661 atccacgcga gacggagccc tcctcgccgg ccggcctgga cgcttgggat ctggttcctg
      721 ttctggggat gtatcgtcag ctctgtatgg agtcaattgt cttctaatgt agcttcctcc
      781 tcctccacct cttcctcgcc ggggtctcac tacccatacg atgtgccaga ttacgccaac
      841 cccacggagc cgggaatcag acgggttccg ggggcctcag aggtgatccg ggagtcgagc
      901 agcacaacag ggatggtcgt cggcattgtg gctgctgccg ccctctgcat cttgatcctc
      961 ctgtacgcca tgtacaagta caggaacagg gacgagggct cgggctcgac ctcgggctcg
     1021 ggcagcggtg gctctggagg ttctggtggc agctctggag gtatgaatgg agctatagga
     1081 ggtgaccttt tgctcaattt tcctgacatg tcggtcctag agcgccaaag ggctcacctc
     1141 aagtacctca atcccacctt tgattctcct ctcgccggct tctttgccga ttcttcaatg
     1201 attaccggcg gcgagatgga cagctatctt tcgactgccg gtttgaatct tccgatgatg
     1261 tacggtgaga cgacggtgga aggtgattca agactctcaa tttcgccgga aacgacgctt
     1321 gggactggaa atttcaaggc agcgaagttt gatacagaga ctaaggattg taatgaggcg
     1381 gcgaagaaga tgacgatgaa cagagatgac ctagtagaag aaggagaaga agagaagtcg
     1441 aaaataacag agcaaaacaa tgggagcaca aaaagcatca agaagatgaa acacaaagcc
     1501 aagaaagaag agaacaattt ctctaatgat tcatctaaag tgacgaagga attggagaaa
     1561 acggattata ttcatagcgg tagtggcacc gtgcgcgtgc cgattgcggt gggcgaaagc
     1621 gattttgaaa acctgaacac cgaagatgtg agcagcgaaa gcgatccggg tggctctggt
     1681 agcttcaacg ctcgcaggaa gctggctggt gctatcctgt tcaccatgct cgccacaaga
     1741 aacttcagcg gcagctttaa tgcgcggcgc aagctggccg gagccatact tttcacaatg
     1801 ttggctacca gaaatttttc ggagctggcc gagaagctgg ccggcctgga catcaacggt
     1861 ggagctagcg agtttcgggc aaccacactg gaacggatcg agaaaagctt cgtgattact
     1921 gatccgagac tgcctgacaa cccaatcatt tttgtgagcg attccttcct gcagctgaca
     1981 gaatattctc gggaagagat cctggggcgc aattgccgtt ttctgcaggg acccgagaca
     2041 gaccgtgcca ctgttcggaa aatcagagat gctattgaca accagactga agtgaccgtt
     2101 cagctgatca attataccaa gagcggcaag aagttctgga acgtgttcca cctgcagccg
     2161 atgcgcgatt ataagggcga cgtccagtac ttcattggcg tgcagctgga tggcaccgaa
     2221 cgtcttcatg gcgccgctga gcgtgaggcg gtctgcctgg tcaaaaagac agcctttcag
     2281 attgctgaga acctgtactt ccagatgggc ggaggatccg actacaagga tgacgacgat
     2341 aagatgtcta gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag
     2401 gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct
     2461 acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg
     2521 ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta
     2581 cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta
     2641 catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt
     2701 ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat
     2761 tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa
     2821 acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac
     2881 caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa
     2941 caacttaaat gtgaaagtgg gtccgcgtac agccgcgcgc gtacgaaaaa caattacggg
     3001 tctaccatcg agggcctgct cgatctcccg gacgacgacg cccccgaaga ggcggggctg
     3061 gcggctccgc gcctgtcctt tctccccgcg ggacacacgc gcagactgtc gacggccccc
     3121 ccgaccgatg tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg
     3181 catgccgacg cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt
     3241 ccgggattta ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag
     3301 tttgagcaga tgtttaccga tgcccttgga attgacgagt acggtgggta ataggaattc
     3361 ctcgagctag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3421 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3481 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3541 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggagg
     3601 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     3661 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     3721 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     3781 gtattttctc cttacgcatc tgtgcggtat tcacaccgca tacgtcaaag caaccatagt
     3841 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3901 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3961 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4021 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4081 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4141 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     4201 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4261 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     4321 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     4381 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4441 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4501 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4561 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4621 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4681 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4741 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4801 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4861 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4921 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4981 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5041 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5101 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5161 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5221 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     5281 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     5341 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa
     5401 gccgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5461 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5521 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5581 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5641 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5701 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5761 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5821 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     5881 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5941 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6001 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6061 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6121 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6181 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6241 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6301 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6361 tgt
//
