LOCUS       sequence-156255-        7193 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of NHA-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 7193)
  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 7193)
  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-1477
FEATURES             Location/Qualifiers
     source          1..7193
                     /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             1096..1161
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     CDS             1189..1215
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2898..3019
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2909..2928)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2963..2982
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      3205..3660
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3292..3311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3502..3523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3777..4134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3777..3968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3800..3820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3985..4120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4185..4979
                     /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(4239..4258)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4849..4868
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5043..5091
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(5132..5151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5251..5273
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5311..5329)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5397..5501
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5502..5570,5571..6362)
                     /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(5720..5739)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6533..7121
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7022..7041
                     /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 ccaccatgga cgcacaaaca cgacgacgtg agcgtcgcgc tgagaaacaa
     1141 gctcaatgga aagctgcaaa cggtggatct cctccaagct tgatggctta cccatacgat
     1201 gttccagatt acgctcctgc aggcatggcc ccacagtcca ggctgaccca gtggaaacaa
     1261 ccactcctcc ctgatcccga gacctctcat ggcgaaacat ggggcatgcc caagattgag
     1321 cagatgtctc tgctgtcagg acctgtagtg agctccagcc tggaacagca gcatggggct
     1381 cccaaccttt ctccacaggc aaataggatt gccgatcccg tgagctcacg gtggggcgtt
     1441 gacgcctctc ttaagctcag cgccgaccct ccagagttca agcccggagt cccctggcgc
     1501 cctcgggggg agggggaaga aaagtctggt tcatctggaa gtagtatgga atcatcccct
     1561 cagggcatgc ctgccagtgg caagactcag ataacagaca acccaaacaa gcagccgggc
     1621 tttccaggcg ggtttagcag cgctagccag ttcccgtact ctatagccag cagtccctgg
     1681 cagaacactg aacccacaga tagcctgaac aagacccagg cgaaccccag tagtatcaga
     1741 ccacctccag ggctcaatac atctactagt agcggcctcc ccgatacccc ctctgtcgaa
     1801 gagagacctg catggatgaa aaatctcatg gatgggggga cccagcagag ctttgcccag
     1861 ggacctaatc ctgggaactt tcagctggga ctgcggttgg gcttctccgc cagcggcaat
     1921 tcctggagta cccaggacgg gatgtctgtt tccggactga ctcctaatag ccaaccgtgg
     1981 gctgccccag gcggacctag ttcttcctcc acaccatcct ctgacccctc tgccgaggcc
     2041 gccagctcca ccgccaatgc cgtaacaagt gctgaacagc ttaaacctca ggatgaaggg
     2101 atctcaagtt ggtccaccaa ccagccaatt ccgtctgcgg aggagaaaca acagcagcca
     2161 cctgtggccc aaggacctag gatgggatcc ggtcgcggcg gagccaaccc aatgagtact
     2221 tggctggtgt tgcggaatct gtccccacgg gcagatccga ccgcaatgag ggctgtgtgc
     2281 cagcagtacg gcccactcct cacctttact ctgaacctta ggcacggaaa cagcttgatc
     2341 agatactcta ataaggatca agccgcctca gcacggaaca acctcaacgg gatgatggtg
     2401 aaaggtatgc agcttatcgc cgactttgcg acggatagcg atattggggg gttttttgag
     2461 cagacacccg actggtccaa caacccaatg ccacccaacc ctagcttcgg caacccttgg
     2521 tccttcggaa cagttgatcc taatcgacct ctcggatcca ctcaaggact ggagaaaacg
     2581 ccgcaaggcc ccccacctca aatgcctgca atggttccgc cgggcatgca atggggaagt
     2641 agttctcagc tccccgctca actctggggc aactcccatc ctggtgccgc cacaggcttc
     2701 ccgtctcagg tgccatccat gtggtctttc agcggaggag cccctcgcga tgtcgaaccc
     2761 caatccggaa caaatgagaa tgggttggtc tctccatcca tgactacgtt tctgcctccc
     2821 gggttgctga acggtggtga gagtgtctaa ggcggccgct tccctttagt gagggttaat
     2881 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     2941 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3001 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3061 ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatccgata
     3121 aggatcgatc cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     3181 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3241 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3301 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3361 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3421 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3481 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3541 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3601 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3661 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3721 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3781 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3841 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3901 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3961 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4021 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4081 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
     4141 gattcttctg acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg
     4201 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4261 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4321 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     4381 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4441 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4501 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4561 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4621 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4681 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     4741 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4801 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     4861 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     4921 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     4981 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggcc
     5041 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     5101 cgataaggat ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5161 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     5221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     5281 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     5341 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     5401 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5461 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     5521 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5581 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5641 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5701 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5761 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5821 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5881 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5941 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6001 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6061 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     6661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7141 ttttacggtt cctggccttt tgctggcctt ttgctcacat ggctcgacag atc
//
