LOCUS       sequence-372429-        7248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express His-tagged NEDD4L C2-W1-L-W2-HECT chimera in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7248)
  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 7248)
  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: pFastBacHTA-NEDD4L_C2-W1-L-W2-HECT
FEATURES             Location/Qualifiers
     source          1..7248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2360..2377
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2511..2735
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2802..3335)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3524..3529,3530..3546,3547..3552))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3904..3995
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3920..3937
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             4062..4079
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4101..4121
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     polyA_signal    6738..6872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6775..6794)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6829..6848
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  6901..7066
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     2281 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     2341 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg
     2401 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagaccagc cgcgtaacct
     2461 ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga
     2521 caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag
     2581 acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt
     2641 tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga
     2701 ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac
     2761 aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg
     2821 tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg
     2881 ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca
     2941 tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact
     3001 gcgagatcat agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc
     3061 gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta
     3121 cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct
     3181 ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg
     3241 agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg
     3301 ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca
     3361 tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa
     3421 acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa
     3481 ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca
     3541 ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac
     3601 cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc
     3661 ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
     3721 cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt
     3781 ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt
     3841 gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa
     3901 tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt
     3961 ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca
     4021 ccatcgggcg cggatctcgg tccgaaacca tgtcgtacta ccatcaccat caccatcacg
     4081 attacgatat cccaacgacc gaaaacctgt attttcaggg catggcgacc gggctcgggg
     4141 agccggtcta tggactttcc gaagacgagg gagagtcccg tattctcaga gtaaaagttg
     4201 tttctggaat tgatctcgcc aaaaaggaca tctttggagc cagtgatccg tatgtgaaac
     4261 tttcattgta cgtagcggat gagaatagag aacttgcttt ggtccagaca aaaacaatta
     4321 aaaagacact gaacccaaaa tggaatgaag aattttattt cagggtaaac ccatctaatc
     4381 acagactcct atttgaagta tttgacgaaa atagactgac acgagacgac ttcctgggcc
     4441 aggtggacgt gccccttagt caccttccga cagaagatcc aaccatggag cgaccctata
     4501 catttaagga ctttctcctc agaccaagaa gtcataagtc tcgagttaag ggatttttgc
     4561 gattgaaaat ggcctatatg ccaaaaaatg gaggtcaaga tgaagaaaac agtgaccaga
     4621 gggatgacat ggagcatgga tgggaagttg ttgactcaaa tgactcggct tctcagcacc
     4681 aagaggaact tcctcctcct cctctgcctc ccgggtggga agaaaaagtg gacaatttag
     4741 gccgaactta ctatgtcaac cacaacaacc ggaccactca gtggcacaga ccaagcctga
     4801 tggacgtgtc ctcggagtcg gacaataaca tcagacagat caaccaggag gcagcacacc
     4861 ggcgcttccg ctcccgcagg cacatcagcg aagacttgga gcccgagccc tcggagggcg
     4921 gggatgtccc cgagccttgg gagaccattt cagaggaagt gaatatcgct ggagactctc
     4981 tcggtctggc tctgccccca ccaccggcct ccccaggatc tcggaccagc cctcaggagc
     5041 tgtcagagga actaagcaga aggcttcaga tcactccaga ctccaatggg gaacagttca
     5101 gctctttgat tcaaagagaa ccctcctcaa ggttgaggtc atgcagtgtc accgacgcag
     5161 ttgcagaaca gggccatcta ccaccgccca gtgccccagc tgggagagcg cgttcatcaa
     5221 ctgtcacggg tggtgaggaa ccaacgccat cagtggccta tgtacatact acgccgggtc
     5281 tgccttcagg ctgggaagaa agaaaagatg ctaaggggcg cacatactat gtcaatcata
     5341 acaatcgaac cacaacttgg actcgaccta tcatgcagaa cccagctatt actggtccgg
     5401 ctgtccctta ctccagagaa tttaagcaga aatatgacta cttcaggaag aaattaaaga
     5461 aacctgctga tatccccaat aggtttgaaa tgaaacttca cagaaataac atatttgaag
     5521 agtcctatcg gagaattatg tccgtgaaaa gaccagatgt cctaaaagct agactgtgga
     5581 ttgagtttga atcagagaaa ggtcttgact atgggggtgt ggccagagaa tggttcttct
     5641 tactgtccaa agagatgttc aacccctact acggcctctt tgagtactct gccacggaca
     5701 actacaccct tcagatcaac cctaattcag gcctctgtaa tgaggatcat ttgtcctact
     5761 tcacttttat tggaagagtt gctggtctgg ccgtatttca tgggaagctc ttagatggtt
     5821 tcttcattag accattttac aagatgatgt tgggaaagca gataaccctg aatgacatgg
     5881 aatctgtgga tagtgaatat tacaactctt tgaaatggat cctggagaat gaccctactg
     5941 agctggacct catgttctgc atagacgaag aaaactttgg acagacatat caagtggatt
     6001 tgaagcccaa tgggtcagaa ataatggtca caaatgaaaa caaaagggaa tatatcgact
     6061 tagtcatcca gtggagattt gtgaacaggg tccagaagca gatgaacgcc ttcttggagg
     6121 gattcacaga actacttcct attgatttga ttaaaatttt tgatgaaaat gagctggagt
     6181 tgctcatgtg cggcctcggt gatgtggatg tgaatgactg gagacagcat tctatttaca
     6241 agaacggcta ctgcccaaac caccccgtca ttcagtggtt ctggaaggct gtgctactca
     6301 tggacgccga aaagcgtatc cggttactgc agtttgtcac agggacatcg cgagtaccta
     6361 tgaatggatt tgccgaactt tatggttcca atggtcctca gctatttaca atagagcaat
     6421 ggggcagtcc tgagaaactg cccagagctc acacatgctt taatcgcctt gacttacctc
     6481 catatgaaac ctttgaagat ttacgagaga aacttctcat ggccgtggaa aatgctcaag
     6541 gatttgaagg ggtggattaa gcggccgctt tcgaatctag agcctgcagt ctcgaggcat
     6601 gcggtaccaa gcttgtcgag aagtactaga ggatcataat cagccatacc acatttgtag
     6661 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     6721 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6781 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6841 aactcatcaa tgtatcttat catgtctgga tctgatcact gcttgagcct aggagatccg
     6901 aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt cgtattagct
     6961 tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa tccttaaaaa
     7021 ctccatttcc acccctccca gttcccaact attttgtccg cccacagcgg ggcatttttc
     7081 ttcctgttat gtttttaatc aaacatcctg ccaactccat gtgacaaacc gtcatcttcg
     7141 gctacttttt ctctgtcaca gaatgaaaat ttttctgtca tctcttcgtt attaatgttt
     7201 gtaattgact gaatatcaac gcttatttgc agcctgaatg gcgaatgg
//
