LOCUS       sequence-357899-        6198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For mouse Nav1.2 knockin with HaloTag-V5  at the C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6198)
  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 6198)
  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-mScn2a-HaloTag-V5-EF1-sfGFP
FEATURES             Location/Qualifiers
     source          1..6198
                     /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..566
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             582..1466
                     /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             1467..1508
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     promoter        1664..1875
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             1888..2156
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             2182..2892
                     /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(2209..2237)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2832..2852
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    2945..3421
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3144..3163)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3461..3601
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3461..3590
                     /label=AAV2 ITR
     rep_origin      3676..4131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3763..3782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3973..3994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4148..4167)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4267..4289
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4327..4345)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4413..4517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4518..4586,4587..5378)
                     /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(4736..4755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5549..6137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6038..6057
                     /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 accggacaag gggaaagata tcagttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag taccggtccc tgatatcttt ccccttgtcc ggttctggat
      541 cgggtgagga tctgtacttt cagagcgata acgatggatc cgaaatcggt actggctttc
      601 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
      661 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
      721 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
      781 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
      841 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
      901 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
      961 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     1021 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     1081 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     1141 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     1201 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     1261 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     1321 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     1381 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     1441 ggctgtctac tctggagatt tccggtggga agcctatacc caatcctctc ctggggttgg
     1501 acagcaccgg ctcaggtagc ggctccctga tatctttccc cttgtcctct agaccaagga
     1561 tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga
     1621 agttgggggg aggggtcggc aattcgctcc ggtgcccgtc agtgggcaga gcgcacatcg
     1681 cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaacgggtgc ctagagaagg
     1741 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     1801 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     1861 gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg cgcccgccgc
     1921 cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc cgcctgtggt
     1981 gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag accgggcctt
     2041 tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct ttgcctgacc
     2101 ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta cagatccaag
     2161 ctgtgaccgg gtaccgccac catgaaagga gaagaacttt tcactggagt tgtcccaatt
     2221 cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtccgtgg agagggtgaa
     2281 ggtgatgcta caaacggaaa actcaccctt aaatttattt gcactactgg aaaactacct
     2341 gttccgtggc caacacttgt cactactctg acctatggtg ttcaatgctt ttcccgttat
     2401 ccggatcaca tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag
     2461 gaacgcacta tatctttcaa agatgacggg acctacaaga cgcgtgctga agtcaagttt
     2521 gaaggtgata cccttgttaa tcgtatcgag ttaaagggta ttgattttaa agaagatgga
     2581 aacattcttg gacacaaact cgagtacaac tttaactcac acaatgtata catcacggca
     2641 gacaaacaaa agaatggaat caaagctaac ttcaaaattc gccacaacgt tgaagatggt
     2701 tccgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt
     2761 ttaccagaca accattacct gtcgacacaa tctgtccttt cgaaagatcc caacgaaaag
     2821 cgtgaccaca tggtccttct tgagtttgta actgctgctg ggattacaca tggcatggat
     2881 gagctctaca aataagaatt cgatatcaag cttatcgata ccgagcgctg ctcgagagat
     2941 ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     3001 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     3061 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     3121 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     3181 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     3241 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     3301 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     3361 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     3421 tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct agtgatggag
     3481 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3541 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3601 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3661 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3721 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     3781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4021 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4081 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4141 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4201 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4261 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4321 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4381 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4441 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4501 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4561 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4621 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4681 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4741 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4801 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4861 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4921 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4981 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5041 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5101 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5161 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5221 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5281 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5341 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5401 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5461 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6181 ggccttttgc tcacatgt
//
