LOCUS       sequence-479183-        7515 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Flp-dependent expression of LATeNT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7515)
  AUTHORS   Roh H, Kim D, Kim B, Jeon Y, Kim Y, Jacko M, Xu F, Lin C, Won Um J, 
            Ting AY
  TITLE     Light-activated tetanus neurotoxin for conditional proteolysis and 
            inducible synaptic inhibition in vivo
  JOURNAL   bioRxiv 2025.01.27.635161; doi: 10.1101/2025.01.27.635161
REFERENCE   2  (bases 1 to 7515)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.01.27.635161; doi: 10.1101/2025.01.27.635161"
COMMENT     Sequence Label: pAAV-EF1a-fDIO-mCherry-LATeNT
FEATURES             Location/Qualifiers
     source          1..7515
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1210..1243
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(3190..3231)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             complement(3232..3939)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3309..3332)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3507..3526)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3776..3794
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      3936..3945
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3952..3985)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(4149..4169)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4548..4688
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4548..4677
                     /label=AAV2 ITR
     rep_origin      4763..5218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4850..4869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5060..5081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5235..5254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5354..5376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5414..5432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5500..5604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5605..5673,5674..6465)
                     /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(5823..5842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6636..7224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7125..7144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7286..7415
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1201 gactccggag aagttcctat tctctagaaa gtataggaac ttcgcagaat ggtagctgga
     1261 ttgtagctgc tattagcaat atgaaacctc ttagaagttc ctattcttca aaaggtatag
     1321 gaacttcggc gcgccttatc aagcggtacg gttgtacagg ttttcacgga tgttggtcgg
     1381 cgggatgatt tttttgcaca gaccgatcag tttggaaacc agaccggaac cgtcgacgtt
     1441 cctaaaggcg ttagtgttca ccctcatgtt ctggccctta tactcggact tcagatcctt
     1501 ggactctatg ttaaagccct cagtatcgtt atatatagta tcatccagca ggttaggtat
     1561 cttcacagga tcgtggttca tggaaaaata gcttaagcga gtctttatgt taaacttctt
     1621 gcccagctct atctcagtaa aaatctgaaa ggctgtcttt ttgaccaggc agaccgcctc
     1681 acgctcagcg gcgccatgaa ggcgttcggt gccatccagc tgcacgccaa tgaagtactg
     1741 gacgtcgccc ttataatcgc gcatcggctg caggtggaac acgttccaga acttcttgcc
     1801 gctcttggta taattgatca gctgaacggt cacttcagtc tggttgtcaa tagcatctct
     1861 gattttccga acagtggcac ggtctgtctc gggtccctgc agaaaacggc aattgcgccc
     1921 caggatctct tcccgagaat attctgtcag ctgcaggaag gaattgctca caaaaatgat
     1981 tgggttgtca ggcagtctcg gatcagtaat cacgaagctt ttctcgatcc gttccagtgt
     2041 ggttgcccga aactccccgc taccggaccc cccgctgccg ctcccgccat acattatgga
     2101 gttatacagt atctgaaact tatcctcgtt cactatatac tggccgttgg aatccttatc
     2161 aaactgatac ttctgctgat agatctgttt gtaggagtcg atgtcgatgt tcgggtcgtt
     2221 gcaggaggta acctgggaca gtttgttagc gatagctttg tagtcgttca gggttttttc
     2281 gtacaggtcg tttttgatat cgatggagat caggttagcg tcctgaccac cgaaggtgaa
     2341 gagctcttca gcggagatcg ggtaggtgtg ctgcatgtag atttcctgtt tggacgggat
     2401 gatttcgtgg gaggaaacct gcatgccgta cagaccgtgc agaacgtgga tcagttcgtg
     2461 catcagcagc agagccgggt cctggaagta tttggattta ccgatggtca gggaggtgat
     2521 gttttcgata acgttgtcga aggtcggtac gtattccggg cagaaagcca tctgcatgat
     2581 ggaaccgaaa ccgtcacggc acgggaagta gtttttgttg tcaacacgca gaacgatacc
     2641 acgaacttcg tttttgttca gaaccggacc cggaccgaag atgatcaggt tggtcagcat
     2701 agcggatttg gtggtagcac cggacggatc ctgttccagc aggttgaagg aaacggagtt
     2761 ggagttggtg tcgaatttgt ccagcaggga gtaggagtta cccaggtacg ggatagcgtt
     2821 gatgattttg tccagcagag cttcaccagc aacgttgttt ttgatacggt tgaacagttt
     2881 aaccatggtc tgcaggaaac ggtctttgtc ggagtcggta cgcaggtagt tcgggtcgta
     2941 gtattcggaa gcaccttcga tcagggagga cggcgggttg aagtcttccg gtttggtacc
     3001 aaattcgtaa cgttccggaa cgatccagat acggtcggtg attttgaagg ctctgtagta
     3061 gatgtctaga cctttgcagt acggcggttc catcatgatg atggtgtcgt tgttaaccgg
     3121 gtcggagtaa cggaagttgt tgatggtgat cggaccggtg cccgagcccg aggtcgagcc
     3181 cgagcccttg gtgctgtcca ggcccagcag ggggttgggg atgggcttgc ccttgtacag
     3241 ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgc tcgtactgtt ccacgatggt
     3301 gtagtcctcg ttgtgggagg tgatgtccag cttgatgttg acgttgtagg cgccgggcag
     3361 ctgcacgggc ttcttggcct tgtaggtggt cttgacctcg gcgtcgtagt ggccgccgtc
     3421 cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg ggtacatccg
     3481 ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt cggaggggaa
     3541 gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcaggg aggagtcctg
     3601 ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact tgaagccctc
     3661 ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt aggccttgga
     3721 gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc cgcccttggt
     3781 caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct cgccctcgat
     3841 ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca tgaactcctt
     3901 gatgatggcc atgttgtcct cctcgccctt gctcaccatg gtggcgctag cgaagttcct
     3961 atactttcta gagaatagga acttcttgcc ttaacccaga aattatcact gttattcttt
     4021 agaatggtgc aaagagaagt tcctatacct tttgaagaat aggaacttcg aattcgatat
     4081 caagcttatc gatataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4141 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4201 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     4261 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4321 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     4381 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4441 ctgcaataaa caagttaaca acaacattgc attcatttta tgtttcaggt tcagggggag
     4501 atgtgggact gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     4561 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     4621 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4681 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4741 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4801 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     4861 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4921 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4981 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     5041 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     5101 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     5161 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     5221 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     5281 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     5341 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5401 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     5461 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5521 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5581 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5641 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5701 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5761 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5821 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5881 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5941 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6001 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6061 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6121 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6181 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6241 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6301 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6361 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6421 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6481 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6541 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6601 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6661 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6721 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6781 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6841 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6901 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6961 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7021 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7081 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7141 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7201 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7261 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
     7321 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     7381 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgta agctttgcaa
     7441 agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt ctaggtcttg
     7501 aaaggagtgg gaatt
//
