LOCUS       sequence-156258-        9043 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of NHA-tagged N-terminal region of 
            Nematostella vectensis GW182 (aa 1-1158) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9043)
  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 9043)
  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-1476
FEATURES             Location/Qualifiers
     source          1..9043
                     /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    4748..4869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4759..4778)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4813..4832
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      5055..5510
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5142..5161)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5352..5373
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5627..5984
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5627..5818
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5650..5670)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5835..5970
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             6035..6829
                     /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(6089..6108)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6699..6718
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6893..6941
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6982..7001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7101..7123
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7161..7179)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7247..7351
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7352..7420,7421..8212)
                     /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(7570..7589)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8383..8971
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8872..8891
                     /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 aggcatgcct aaggatgaca agaaggaatt cctcaagtca
     1261 ttgggcgttg acaccctgtc ctctgaaaac gacatcaaga gagcctataa gaaaatagca
     1321 ctccagtgtc accctgataa gaaccccagc gacacgtccg ccagaaaaca attcgaggag
     1381 gtctctaaag cattcgagta cctcgtccac gacgggatca atgacagcct gaaagattac
     1441 gataaggttt tctttgattt cctccgggac atattgttcg gcagcgatgg tgcttcagac
     1501 ttctttacct atttttgtgg ttccaagcag cacagctgta tctccacaga caccgaccct
     1561 gattttagat atcaggatga cgacgaggag gaggacattg atggggaaca ggagctggaa
     1621 agcctcctgg agatcgtcaa gaaccgccgg aagcgcggga agcccgtacc cgatgagttt
     1681 cacgaggaga aactgcagga gctgaagact cgactgtctc agcacaaaga ggacatgaaa
     1741 aaggatggga gcaagaaaca ggctgttaag aaggggaaag ctgtcagcgt ccctgtggca
     1801 aaacccaagc caaagagcaa aaagcagctc cttgctgaac aacggcggcg ggctaaagaa
     1861 atggagcaga tagccatgga actggaggag aaaagaaaag aagaagaaga aaaggagaag
     1921 caacgcaaac agctggaagt agagaaacag aaacgccttg aggaggagcg catcaggtca
     1981 cagaacgagg agcgaaaacg gcgggagcga gagcaaaaag aacgggagaa gcagcagaaa
     2041 ctggatatgg agaaagagga gcgccgccgg cgccagcagg aggaggagaa gaaaagacga
     2101 gaggaggaag agaagagaaa aaaagtcgag aaagagaagc aggaaaggga gaaggccgca
     2161 aaaaaagctg ccctccaggc tgcgaaggag aaagaagaac aggcgaaggc tgcatctaaa
     2221 aatcagaata ttaaaagaca gcagcaacaa cagcgaactg atcaacagca gtatcctagg
     2281 gaaatacctc ctaggtttca gaaaatgaaa agacagcaac aacagcagca gctgcagact
     2341 ccaaacttgc cacctccagc cccaaccggc tctccggctc agcttaacaa gttccagtca
     2401 aaacctcagc aacagcctgt tggcaagaag aaggatccta accgcttcgc tagcaaggag
     2461 tacgctgacc tcgttgacga ggtggacgat gcgggctgga acaagggatc tcagtgtaaa
     2521 ggctctggat gggaagggac tagcgaggat tgggaggaga acatcgccga aaagtggggc
     2581 gcagataggc agtgggggag caaggaaggg tccagtaacc aactcgttgt ggaccgcaca
     2641 gatggcgata gctggcccac cgtgggcagt aagtctgcta ttggagataa gcccaattct
     2701 tcccttgaga acagtaacag caaaaacagc agtgagaccg tgcctgtggg gacaatcggc
     2761 agcaaacctc caagcagcaa ttggaacagt aacaggaaca gcggaggact cccactgtcc
     2821 gagaattggg atacaacctg tgacgagagc atcgatcgcg ctctggacgc cactgaatct
     2881 aaaggatcag tggggcctgg gggagtcaat tgggttggca tgggtacagg cggattgggc
     2941 gggatcattg gaaccccttt gggttgggag cctagcaaag agaaccgcga tattgataag
     3001 accgcctcta aaagcggctg gtgcggggaa cagaactcta ctgtcgatag ctggaataag
     3061 actccaggta agggcccgaa aacacaggtg ttcggtggat cagcccccac atggggggga
     3121 acaccggcag actcttcttc cgcctgggtg gagactagcg ggactgagac caataaaccc
     3181 aactcctccg gctggaatag caaggtcagc agcggcctga gtcagggaaa ctctagtagc
     3241 atggcctctg tagatagtac cgacaataaa ccaacagaca ataaaccagc tccatcaggc
     3301 tggggcgaag ccggcccatc atgggctgtg caggcttccg ttaccggaac agcatgctgg
     3361 gacaccgcca ctaccacatc ccctaaaaag cctctggatt ccaaagatag caagctgagc
     3421 cacgataagg tgtccgggtg gctcgagaca tccgtgacct ccactagcgt gactgcagat
     3481 tggaacggcg atagcaatgc tgaccaggag gactactacg aagaggcgtg ggacgggtgg
     3541 acaactgcat cctccaagcg aaacaaggcc ccgaagccgg cacccacaaa tgactccaat
     3601 gcatggatgt ccaggagcct taaacagctt ctggacatgg gatttaaaca agaggacgct
     3661 gagaaagcgc tgcgcgccaa tcacatgaac atcgagagcg cgatttctga cctgctgagc
     3721 cttacagcct cccagatcga ggaagcctcc cgagctagtt ctgctaacac cggtgagcct
     3781 gacccggcag aagcctgtag gatgcagcag cagaatacta ctacatctaa gccaaaccga
     3841 aaacaaaggc gcaagaccca gcagcagttg cagcaacagc aggcctctca ggccaagcag
     3901 gagtccaagc aaaccgatat ctacgatgct aatagtcagt cagggtcttc tgactctaca
     3961 gttaaggatc ccaaatctga gtccaataaa gataaagaag gccaggccgc cgagggccag
     4021 agtaataagc cttctaatca gggggctagc gacgggaaag ggaacgcctc aaaaaccaca
     4081 gaaaattctc cacacctctc accgggtaag tccaatgcag gtcagaaacg agccacaact
     4141 ggcctcatcc agacgcctgc attggggacc ccgaccccaa tgagaactcc gatgacacat
     4201 cctttgttgc accagcagtt tctgcatcag gtgcagctcc accagctgag gatggcccag
     4261 ggaggtctcc ttcaacctcc cataaacccc cagcaagtag ctttgttgca gcagatggct
     4321 cagcttcaga tggtccagca tcgcatcgcc cagcagcaac tgacccagaa gcacaacatc
     4381 caggcacaac agcagctgta tcaacagcag caacagattg cactcatgat ggggcagctg
     4441 cagcagcaag tgatgcagca gcacaatcat cagatagccg gttcccggtt ccccagcccc
     4501 ggctttcagc agagcaccct tcctcctcaa agcagttccg cccagcctgg tactccacct
     4561 cagaacgcca agaattccac caaagccact tctactgagc ctagtcaatc aaaacctgag
     4621 gaacccccca aggtgtccac cgcagataag cctgtgacca cagttgccag cggagagcga
     4681 ataactcagc agagcccttg agcggccgct tccctttagt gagggttaat gcttcgagca
     4741 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4801 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4861 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg
     4921 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatccgata aggatcgatc
     4981 cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5041 tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5101 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5161 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5221 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5281 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5341 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5401 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5461 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt cctgatgcgg
     5521 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc tgcgcagcac
     5581 catggcctga aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga
     5641 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5701 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5761 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5821 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5881 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     5941 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt gattcttctg
     6001 acacaacagt ctcgaactta aggctagagc caccatgatt gaacaagatg gattgcacgc
     6061 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     6121 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     6181 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     6241 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     6301 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     6361 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     6421 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     6481 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     6541 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     6601 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     6661 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     6721 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     6781 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     6841 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgatggcc gcaataaaat
     6901 atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag cgataaggat
     6961 ccgcgtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     7021 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     7081 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     7141 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     7201 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     7261 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     7321 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     7381 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     7441 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     7501 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     7561 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     7621 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     7681 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7741 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7801 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7861 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7921 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7981 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     8041 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     8101 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     8161 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     8221 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     8281 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8341 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     8401 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     8461 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     8521 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     8581 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     8641 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     8701 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     8761 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     8821 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     8881 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     8941 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     9001 cctggccttt tgctggcctt ttgctcacat ggctcgacag atc
//
