LOCUS       sequence-156262-        7106 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of HA-tagged C-terminal region of 
            Nematostella vectensis GW182 (aa 1159-1698) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7106)
  AUTHORS   Mauri M, Kirchner M, Aharoni R, Ciolli Mattioli C, van den Bruck D, 
            Gutkovitch N, Modepalli V, Selbach M, Moran Y, Chekulaeva M
  TITLE     Conservation of miRNA-mediated silencing mechanisms across 600 
            million years of animal evolution.
  JOURNAL   Nucleic Acids Res. 2016 Sep 6. pii: gkw792.
  PUBMED    27604873
REFERENCE   2  (bases 1 to 7106)
  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: "Nucleic 
            Acids Res. 2016 Sep 6. pii: gkw792."
COMMENT     Sequence Label: pMCh-1475
FEATURES             Location/Qualifiers
     source          1..7106
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..518
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        519..730
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     681..701
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     727..751
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          891..1023
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1006..1025
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1068..1086
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1090..1099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1102..1128
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2811..2932
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2822..2841)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2876..2895
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3118..3573
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3205..3224)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3415..3436
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3690..4047
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3690..3881
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3713..3733)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3898..4033
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4098..4892
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4152..4171)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4762..4781
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4956..5004
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5045..5064)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5164..5186
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5224..5242)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5310..5414
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5415..5483,5484..6275)
                     /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(5633..5652)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6446..7034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6935..6954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcaatattg gccattagcc atattattca ttggttatat agcataaatc aatattggct
       61 attggccatt gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc
      121 caatatgacc gccatgttgg cattgattat tgactagtta ttaatagtaa tcaattacgg
      181 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      241 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      301 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      361 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
      421 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
      481 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      541 ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      601 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
      661 gcgatcgccc gccccgttga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      721 aagcagagct cgtttagtga accgtcagat cactagaagc tttattgcgg tagtttatca
      781 cagttaaatt gctaacgcag tcagtgcttc tgacacaaca gtctcgaact taagctgcag
      841 tgactctctt aaggtagcct tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca
      901 aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga cagagaagac
      961 tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc tttctctcca
     1021 caggtgtcca ctcccagttc aattacagct cttaaggcta gagtacttaa tacgactcac
     1081 tataggctag ccaccatggc ttacccatac gatgttccag attacgctcc tgcaggcatg
     1141 gccccacagt ccaggctgac ccagtggaaa caaccactcc tccctgatcc cgagacctct
     1201 catggcgaaa catggggcat gcccaagatt gagcagatgt ctctgctgtc aggacctgta
     1261 gtgagctcca gcctggaaca gcagcatggg gctcccaacc tttctccaca ggcaaatagg
     1321 attgccgatc ccgtgagctc acggtggggc gttgacgcct ctcttaagct cagcgccgac
     1381 cctccagagt tcaagcccgg agtcccctgg cgccctcggg gggaggggga agaaaagtct
     1441 ggttcatctg gaagtagtat ggaatcatcc cctcagggca tgcctgccag tggcaagact
     1501 cagataacag acaacccaaa caagcagccg ggctttccag gcgggtttag cagcgctagc
     1561 cagttcccgt actctatagc cagcagtccc tggcagaaca ctgaacccac agatagcctg
     1621 aacaagaccc aggcgaaccc cagtagtatc agaccacctc cagggctcaa tacatctact
     1681 agtagcggcc tccccgatac cccctctgtc gaagagagac ctgcatggat gaaaaatctc
     1741 atggatgggg ggacccagca gagctttgcc cagggaccta atcctgggaa ctttcagctg
     1801 ggactgcggt tgggcttctc cgccagcggc aattcctgga gtacccagga cgggatgtct
     1861 gtttccggac tgactcctaa tagccaaccg tgggctgccc caggcggacc tagttcttcc
     1921 tccacaccat cctctgaccc ctctgccgag gccgccagct ccaccgccaa tgccgtaaca
     1981 agtgctgaac agcttaaacc tcaggatgaa gggatctcaa gttggtccac caaccagcca
     2041 attccgtctg cggaggagaa acaacagcag ccacctgtgg cccaaggacc taggatggga
     2101 tccggtcgcg gcggagccaa cccaatgagt acttggctgg tgttgcggaa tctgtcccca
     2161 cgggcagatc cgaccgcaat gagggctgtg tgccagcagt acggcccact cctcaccttt
     2221 actctgaacc ttaggcacgg aaacagcttg atcagatact ctaataagga tcaagccgcc
     2281 tcagcacgga acaacctcaa cgggatgatg gtgaaaggta tgcagcttat cgccgacttt
     2341 gcgacggata gcgatattgg ggggtttttt gagcagacac ccgactggtc caacaaccca
     2401 atgccaccca accctagctt cggcaaccct tggtccttcg gaacagttga tcctaatcga
     2461 cctctcggat ccactcaagg actggagaaa acgccgcaag gccccccacc tcaaatgcct
     2521 gcaatggttc cgccgggcat gcaatgggga agtagttctc agctccccgc tcaactctgg
     2581 ggcaactccc atcctggtgc cgccacaggc ttcccgtctc aggtgccatc catgtggtct
     2641 ttcagcggag gagcccctcg cgatgtcgaa ccccaatccg gaacaaatga gaatgggttg
     2701 gtctctccat ccatgactac gtttctgcct cccgggttgc tgaacggtgg tgagagtgtc
     2761 taaggcggcc gcttcccttt agtgagggtt aatgcttcga gcagacatga taagatacat
     2821 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     2881 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     2941 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa
     3001 gtaaaacctc tacaaatgtg gtaaaatccg ataaggatcg atccgggctg gcgtaatagc
     3061 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg
     3121 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3181 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3241 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3301 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3361 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3421 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3481 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3541 cgaattttaa caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca
     3601 tctgtgcggt atttcacacc gcatacgcgg atctgcgcag caccatggcc tgaaataacc
     3661 tctgaaagag gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg
     3721 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3781 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     3841 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3901 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3961 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4021 ttttttggag gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac
     4081 ttaaggctag agccaccatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4141 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4201 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4261 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4321 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4381 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4441 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4501 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4561 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4621 aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4681 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     4741 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     4801 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     4861 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     4921 ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta ttttcattac
     4981 atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatccgcgta tggtgcactc
     5041 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5101 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5161 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5221 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5281 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5341 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5401 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5461 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5521 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5581 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5641 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5701 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5761 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5821 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5881 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5941 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6001 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6061 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6121 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6181 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6241 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6301 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6361 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6421 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6481 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6541 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6601 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6661 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6721 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     6781 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     6841 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     6901 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     6961 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7021 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7081 cttttgctca catggctcga cagatc
//
