LOCUS       sequence-372456-        6331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express a chimera of the Nav1.5 linker between the first and second 
            transmembrane domain with the motif surrounding PPSY; residues 
            1974-1977 PPSY mutated to AASA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6331)
  AUTHORS   Wright KM, Nathan S, Jiang H, Xia W, Kim H, Chakouri N, Nwafor JN, 
            Fossier L, Srinivasan L, Chen Z, Boronina T, Post J, Paul S, Cole 
            RN, Ben-Johny M, Cole PA, Gabelli SB
  TITLE     NEDD4L intramolecular interactions regulate its auto and substrate 
            Na(V)1.5 ubiquitination.
  JOURNAL   J Biol Chem. 2024 Mar;300(3):105715. doi: 10.1016/j.jbc.2024.105715.
            Epub 2024 Feb 2.
  PUBMED    38309503
REFERENCE   2  (bases 1 to 6331)
  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: "J Biol 
            Chem."; date: "2024-03"; volume: "300(3):105715. doi"; pages: " 
            10.1016/j.jbc.2024.105715. Epub 2024 Feb 2"
COMMENT     Sequence Label: Nav1.5_1-2linker411-717_PPSYtoAASA1960-1996
FEATURES             Location/Qualifiers
     source          1..6331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             45..62
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             87..107
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1149..1166
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1219..1237)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1233..1280
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1317..1772
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1404..1423)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1614..1635
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1864..2679)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1864..2679)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2587..2606
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2671..2690
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2801..3389
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3290..3309
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3543..3560
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3575..3714
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3640..3659)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3800..3822
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3816..4007)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4816..5898)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5860..5879
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5899..5976)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6182..6201
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6285..6304
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6285..6303
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6304..6328
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
       61 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccatg gaggaacaga
      121 accaagcgac cattgcggag accgaggaga aggagaagcg ttttcaagag gcgatggaaa
      181 tgctgaagaa agagcacgaa gcgctgacca tccgtggtgt ggacaccgtt agccgtagca
      241 gcctggagat gagcccgctg gcgccggtga acagccatga acgtcgtagc aagcgtcgta
      301 aacgtatgag cagcggtacc gaggaatgcg gcgaggatcg tctgccgaag agcgacagcg
      361 aagatggtcc gcgtgcgatg aaccatctga gcctgacccg tggcctgagc cgtaccagca
      421 tgaaaccgcg tagcagccgt ggtagcattt tcacctttcg tcgtcgtgac ctgggcagcg
      481 aggcggattt cgcggacgat gaaaacagca ccgcgggcga gagcgaaagc caccatacca
      541 gcctgctggt tccgtggccg ctgcgtcgta ccagcgcgca gggtcaaccg agcccgggta
      601 ccagcgcgcc gggtcatgcg ctgcacggca agaaaaacag caccgtggac tgcaacggtg
      661 tggttagcct gctgggtgcg ggcgatccgg aggcgaccag cccgggcagc cacctgctgc
      721 gtccggttat gctggaacac ccgccggaca ccaccacccc gagcgaggaa ccgggtggcc
      781 cgcagatgct gaccagccaa gcgccgtgcg tggatggttt cgaggaaccg ggtgcgcgtc
      841 agcgtgcgct gagcgcggtg agcgttctga ccagcgcgct ggaggaactg gaggaaagcc
      901 gtcacaagtg cccgccgtgc tggaaccgtc tggcgcagcg ttacctgatc tgggagtgct
      961 gcccgctgtg gatgagcatt aagcaaggtg ttaaactggt ggttatggac ccgctgggtc
     1021 cgccgagcag cagcagcatc agcagcacca gctttgcggc gagcgctgac agcgttaccc
     1081 gtgcgaccag cgacaatctg caagtgcgtg gcagcgacta cagctaaaag cttgcggccg
     1141 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1201 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1261 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1321 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1381 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1441 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1501 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1561 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1621 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1681 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1741 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     1801 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1861 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1921 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1981 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     2041 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     2101 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     2161 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     2221 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     2281 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2341 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     2401 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2461 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2521 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2581 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2641 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     2701 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2761 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2821 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2881 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2941 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3001 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3061 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3121 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3181 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3241 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3301 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3361 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3421 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3481 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3541 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3601 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3661 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3721 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3781 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3841 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3901 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3961 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4021 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4081 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4141 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4201 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4261 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4321 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4381 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4441 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4501 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4561 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4621 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4681 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4741 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4801 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4861 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4921 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4981 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5041 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5101 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5161 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5221 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5281 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5341 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5401 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5461 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5521 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5581 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5641 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5701 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5761 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5821 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5881 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5941 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6001 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6061 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6121 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6181 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6241 cgatgcgtcc ggcgtagagg atcgagatct cgaccccgcg aaattaatac gactcactat
     6301 aggggaattg tgagcggata acaattcccc t
//
