LOCUS       sequence-357891-        6195 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mouse Nav1.6 knockin with HaloTag-V5 at the C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6195)
  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 6195)
  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"; 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-mScn8a-HaloTag-V5-EF1-sfGFP
FEATURES             Location/Qualifiers
     source          1..6195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   9..138
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        153..401
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     153..173
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     324..343
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        422..497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             552..572
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             588..1472
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             1473..1514
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        1669..1880
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1893..2161
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2187..2897
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2214..2242)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2837..2857
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2950..3426
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3149..3168)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3466..3606
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3466..3595
                     /label=AAV2 ITR
     rep_origin      3681..4136
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3768..3787)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3978..3999
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4153..4172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4272..4294
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4332..4350)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4418..4522
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4523..4591,4592..5383)
                     /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(4741..4760)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5554..6142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6043..6062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg
       61 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
      121 ccatcactag gggttcctgc ggccgcacgc gtgagggcct atttcccatg attccttcat
      181 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
      241 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      301 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      361 atttcttggc tttatatatc ttgtggaaag gacgaaacac cccgacaagg agaagcagca
      421 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      481 tggcaccgag tcggtgcttt ttttctagta ccggtccgct gctgcttctc cttgtcggtt
      541 ctggatcggg tgaggatctg tactttcaga gcgataacga tggatccgaa atcggtactg
      601 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
      661 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
      721 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
      781 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
      841 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
      901 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
      961 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     1021 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     1081 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     1141 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     1201 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     1261 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     1321 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     1381 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     1441 cgcgctggct gtctactctg gagatttccg gtgggaagcc tatacccaat cctctcctgg
     1501 ggttggacag caccggctca ggtagcggcc cgctgctgct tctccttgtc ggtctagacc
     1561 aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
     1621 cgagaagttg gggggagggg tcggcaattc gctccggtgc ccgtcagtgg gcagagcgca
     1681 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacg ggtgcctaga
     1741 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     1801 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     1861 ggtttgccgc cagaacacag ctgaagcttc gaggggctcg catctctcct tcacgcgccc
     1921 gccgccctac ctgaggccgc catccacgcc ggttgagtcg cgttctgccg cctcccgcct
     1981 gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt aaagctcagg tcgagaccgg
     2041 gcctttgtcc ggcgctccct tggagcctac ctagactcag ccggctctcc acgctttgcc
     2101 tgaccctgct tgctcaactc tacgtctttg tttcgttttc tgttctgcgc cgttacagat
     2161 ccaagctgtg accgggtacc gccaccatga aaggagaaga acttttcact ggagttgtcc
     2221 caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc cgtggagagg
     2281 gtgaaggtga tgctacaaac ggaaaactca cccttaaatt tatttgcact actggaaaac
     2341 tacctgttcc gtggccaaca cttgtcacta ctctgaccta tggtgttcaa tgcttttccc
     2401 gttatccgga tcacatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     2461 tacaggaacg cactatatct ttcaaagatg acgggaccta caagacgcgt gctgaagtca
     2521 agtttgaagg tgataccctt gttaatcgta tcgagttaaa gggtattgat tttaaagaag
     2581 atggaaacat tcttggacac aaactcgagt acaactttaa ctcacacaat gtatacatca
     2641 cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacgttgaag
     2701 atggttccgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
     2761 tccttttacc agacaaccat tacctgtcga cacaatctgt cctttcgaaa gatcccaacg
     2821 aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca
     2881 tggatgagct ctacaaataa gaattcgata tcaagcttat cgataccgag cgctgctcga
     2941 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     3001 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     3061 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     3121 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     3181 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     3241 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     3301 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     3361 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     3421 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     3481 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3541 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     3601 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3661 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3721 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     3781 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3841 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     3901 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3961 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     4021 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     4081 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     4141 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4201 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4261 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4321 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4381 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4441 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4501 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4561 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4621 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4681 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4741 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4801 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4861 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4921 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4981 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5041 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5101 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5161 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5221 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5281 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5341 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5401 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5461 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5521 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5581 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5641 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5701 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5761 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5821 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5881 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5941 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6001 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6061 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6181 ttgctggcct tttgc
//
