LOCUS       sequence-357910-        5485 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For rat Nav1.2 knockin with 3xV5 at the extracellular loop.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5485)
  AUTHORS   Liu H, Wang HG, Pitt GS, Liu ZJ
  TITLE     Direct Observation of Compartment-Specific Localization and Dynamics
            of Voltage-Gated Sodium Channels.
  JOURNAL   J Neurosci. 2022 Jun 6;42(28):5482-98. doi: 
            10.1523/JNEUROSCI.0086-22.2022.
  PUBMED    35672149
REFERENCE   2  (bases 1 to 5485)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1523/JNEUROSCI.0086-22"; journalName: "J Neurosci"; date: 
            "2022-06-6- 6"; volume: "42"; issue: "28"; pages: "5482-98"
COMMENT     Sequence Label: PX552-rScn2a-3xV5-Syn-miRFP670
FEATURES             Location/Qualifiers
     source          1..5485
                     /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        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             546..587
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             603..644
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             660..701
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        768..1215
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      1229..1238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1235..2182
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    2232..2708
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2431..2450)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2748..2888
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2748..2877
                     /label=AAV2 ITR
     rep_origin      2963..3418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3050..3069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3260..3281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3435..3454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3554..3576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3614..3632)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3700..3804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3805..3873,3874..4665)
                     /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(4023..4042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4836..5424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5325..5344
                     /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 gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac acctggtact gccttcaata ggagttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag taccggtccg tcctattgaa ggcagtacca ggttctggat
      541 cgggtgggaa gcctataccc aatcctctcc tggggttgga cagcaccggc agtggtagcg
      601 gtggcaagcc catccccaac cccctgctgg gcctggactc caccggctcc ggcagtggtg
      661 gaaaacctat tcccaaccca ctgctgggac tggattctac aggctcaggt agcggctccg
      721 tcctattgaa ggcagtacca tctagactgc agagggccct gcgtatgagt gcaagtgggt
      781 tttaggacca ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg
      841 gacaagcacc caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga
      901 aacaggatgc ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg
      961 cccccgcctg gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc
     1021 gccggtcccc cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc
     1081 ccagccggac cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc
     1141 tgcggcgccg gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg
     1201 tgcctgagag cgcagtcgag aaggtaccgc caccatggta gcaggtcatg cctctggcag
     1261 ccccgcattc gggaccgcct ctcattcgaa ttgcgaacat gaagagatcc acctcgccgg
     1321 ctcgatccag ccgcatggcg cgcttctggt cgtcagcgaa catgatcatc gcgtcatcca
     1381 ggccagcgcc aacgccgcgg aatttctgaa tctcggaagc gtactcggcg ttccgctcgc
     1441 cgagatcgac ggcgatctgt tgatcaagat cctgccgcat ctcgatccca ccgccgaagg
     1501 catgccggtc gcggtgcgct gccggatcgg caatccctct acggagtact gcggtctgat
     1561 gcatcggcct ccggaaggcg ggctgatcat cgaactcgaa cgtgccggcc cgtcgatcga
     1621 tctgtcaggc acgctggcgc cggcgctgga gcggatccgc acggcgggtt cactgcgcgc
     1681 gctgtgcgat gacaccgtgc tgctgtttca gcagtgcacc ggctacgacc gggtgatggt
     1741 gtatcgtttc gatgagcaag gccacggcct ggtattctcc gagtgccatg tgcctgggct
     1801 cgaatcctat ttcggcaacc gctatccgtc gtcgactgtc ccgcagatgg cgcggcagct
     1861 gtacgtgcgg cagcgcgtcc gcgtgctggt cgacgtcacc tatcagccgg tgccgctgga
     1921 gccgcggctg tcgccgctga ccgggcgcga tctcgacatg tcgggctgct tcctgcgctc
     1981 gatgtcgccg tgccatctgc agttcctgaa ggacatgggc gtgcgcgcca ccctggcggt
     2041 gtcgctggtg gtcggcggca agctgtgggg cctggttgtc tgtcaccatt atctgccgcg
     2101 cttcatccgt ttcgagctgc gggcgatctg caaacggctc gccgaaagga tcgcgacgcg
     2161 gatcaccgcg cttgagagct aagaattcga tatcaagctt atcgataccg agcgctgctc
     2221 gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg gccctggaag
     2281 ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc attttgtctg
     2341 actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc aaggggcaag
     2401 ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg agtgcagtgg
     2461 cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc ctgcctcagc
     2521 ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt ttgttttttt
     2581 ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa tctcaggtga
     2641 tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact gctcccttcc
     2701 ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     2761 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     2821 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     2881 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2941 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3001 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     3061 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3121 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     3181 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3241 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     3301 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     3361 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     3421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3481 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3541 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3601 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3661 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3721 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3781 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3841 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3901 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3961 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4021 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4081 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4141 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4201 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4261 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4321 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4381 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4441 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4501 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4561 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4621 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4681 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4741 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4801 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4861 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4921 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4981 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5041 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5101 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5161 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5221 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5281 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5341 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5401 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5461 ttttgctggc cttttgctca catgt
//
