LOCUS       sequence-156264-        8956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of HA-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 8956)
  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 8956)
  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-1474
FEATURES             Location/Qualifiers
     source          1..8956
                     /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    4661..4782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4672..4691)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4726..4745
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4968..5423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5055..5074)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5265..5286
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5540..5897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5540..5731
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5563..5583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5748..5883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5948..6742
                     /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(6002..6021)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6612..6631
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6806..6854
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6895..6914)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7014..7036
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7074..7092)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7160..7264
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7265..7333,7334..8125)
                     /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(7483..7502)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8296..8884
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8785..8804
                     /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 cctaaggatg acaagaagga attcctcaag tcattgggcg ttgacaccct gtcctctgaa
     1201 aacgacatca agagagccta taagaaaata gcactccagt gtcaccctga taagaacccc
     1261 agcgacacgt ccgccagaaa acaattcgag gaggtctcta aagcattcga gtacctcgtc
     1321 cacgacggga tcaatgacag cctgaaagat tacgataagg ttttctttga tttcctccgg
     1381 gacatattgt tcggcagcga tggtgcttca gacttcttta cctatttttg tggttccaag
     1441 cagcacagct gtatctccac agacaccgac cctgatttta gatatcagga tgacgacgag
     1501 gaggaggaca ttgatgggga acaggagctg gaaagcctcc tggagatcgt caagaaccgc
     1561 cggaagcgcg ggaagcccgt acccgatgag tttcacgagg agaaactgca ggagctgaag
     1621 actcgactgt ctcagcacaa agaggacatg aaaaaggatg ggagcaagaa acaggctgtt
     1681 aagaagggga aagctgtcag cgtccctgtg gcaaaaccca agccaaagag caaaaagcag
     1741 ctccttgctg aacaacggcg gcgggctaaa gaaatggagc agatagccat ggaactggag
     1801 gagaaaagaa aagaagaaga agaaaaggag aagcaacgca aacagctgga agtagagaaa
     1861 cagaaacgcc ttgaggagga gcgcatcagg tcacagaacg aggagcgaaa acggcgggag
     1921 cgagagcaaa aagaacggga gaagcagcag aaactggata tggagaaaga ggagcgccgc
     1981 cggcgccagc aggaggagga gaagaaaaga cgagaggagg aagagaagag aaaaaaagtc
     2041 gagaaagaga agcaggaaag ggagaaggcc gcaaaaaaag ctgccctcca ggctgcgaag
     2101 gagaaagaag aacaggcgaa ggctgcatct aaaaatcaga atattaaaag acagcagcaa
     2161 caacagcgaa ctgatcaaca gcagtatcct agggaaatac ctcctaggtt tcagaaaatg
     2221 aaaagacagc aacaacagca gcagctgcag actccaaact tgccacctcc agccccaacc
     2281 ggctctccgg ctcagcttaa caagttccag tcaaaacctc agcaacagcc tgttggcaag
     2341 aagaaggatc ctaaccgctt cgctagcaag gagtacgctg acctcgttga cgaggtggac
     2401 gatgcgggct ggaacaaggg atctcagtgt aaaggctctg gatgggaagg gactagcgag
     2461 gattgggagg agaacatcgc cgaaaagtgg ggcgcagata ggcagtgggg gagcaaggaa
     2521 gggtccagta accaactcgt tgtggaccgc acagatggcg atagctggcc caccgtgggc
     2581 agtaagtctg ctattggaga taagcccaat tcttcccttg agaacagtaa cagcaaaaac
     2641 agcagtgaga ccgtgcctgt ggggacaatc ggcagcaaac ctccaagcag caattggaac
     2701 agtaacagga acagcggagg actcccactg tccgagaatt gggatacaac ctgtgacgag
     2761 agcatcgatc gcgctctgga cgccactgaa tctaaaggat cagtggggcc tgggggagtc
     2821 aattgggttg gcatgggtac aggcggattg ggcgggatca ttggaacccc tttgggttgg
     2881 gagcctagca aagagaaccg cgatattgat aagaccgcct ctaaaagcgg ctggtgcggg
     2941 gaacagaact ctactgtcga tagctggaat aagactccag gtaagggccc gaaaacacag
     3001 gtgttcggtg gatcagcccc cacatggggg ggaacaccgg cagactcttc ttccgcctgg
     3061 gtggagacta gcgggactga gaccaataaa cccaactcct ccggctggaa tagcaaggtc
     3121 agcagcggcc tgagtcaggg aaactctagt agcatggcct ctgtagatag taccgacaat
     3181 aaaccaacag acaataaacc agctccatca ggctggggcg aagccggccc atcatgggct
     3241 gtgcaggctt ccgttaccgg aacagcatgc tgggacaccg ccactaccac atcccctaaa
     3301 aagcctctgg attccaaaga tagcaagctg agccacgata aggtgtccgg gtggctcgag
     3361 acatccgtga cctccactag cgtgactgca gattggaacg gcgatagcaa tgctgaccag
     3421 gaggactact acgaagaggc gtgggacggg tggacaactg catcctccaa gcgaaacaag
     3481 gccccgaagc cggcacccac aaatgactcc aatgcatgga tgtccaggag ccttaaacag
     3541 cttctggaca tgggatttaa acaagaggac gctgagaaag cgctgcgcgc caatcacatg
     3601 aacatcgaga gcgcgatttc tgacctgctg agccttacag cctcccagat cgaggaagcc
     3661 tcccgagcta gttctgctaa caccggtgag cctgacccgg cagaagcctg taggatgcag
     3721 cagcagaata ctactacatc taagccaaac cgaaaacaaa ggcgcaagac ccagcagcag
     3781 ttgcagcaac agcaggcctc tcaggccaag caggagtcca agcaaaccga tatctacgat
     3841 gctaatagtc agtcagggtc ttctgactct acagttaagg atcccaaatc tgagtccaat
     3901 aaagataaag aaggccaggc cgccgagggc cagagtaata agccttctaa tcagggggct
     3961 agcgacggga aagggaacgc ctcaaaaacc acagaaaatt ctccacacct ctcaccgggt
     4021 aagtccaatg caggtcagaa acgagccaca actggcctca tccagacgcc tgcattgggg
     4081 accccgaccc caatgagaac tccgatgaca catcctttgt tgcaccagca gtttctgcat
     4141 caggtgcagc tccaccagct gaggatggcc cagggaggtc tccttcaacc tcccataaac
     4201 ccccagcaag tagctttgtt gcagcagatg gctcagcttc agatggtcca gcatcgcatc
     4261 gcccagcagc aactgaccca gaagcacaac atccaggcac aacagcagct gtatcaacag
     4321 cagcaacaga ttgcactcat gatggggcag ctgcagcagc aagtgatgca gcagcacaat
     4381 catcagatag ccggttcccg gttccccagc cccggctttc agcagagcac ccttcctcct
     4441 caaagcagtt ccgcccagcc tggtactcca cctcagaacg ccaagaattc caccaaagcc
     4501 acttctactg agcctagtca atcaaaacct gaggaacccc ccaaggtgtc caccgcagat
     4561 aagcctgtga ccacagttgc cagcggagag cgaataactc agcagagccc ttgagcggcc
     4621 gcttcccttt agtgagggtt aatgcttcga gcagacatga taagatacat tgatgagttt
     4681 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     4741 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     4801 cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc
     4861 tacaaatgtg gtaaaatccg ataaggatcg atccgggctg gcgtaatagc gaagaggccc
     4921 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag
     4981 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5041 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5101 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5161 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5221 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5281 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5341 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5401 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     5461 atttcacacc gcatacgcgg atctgcgcag caccatggcc tgaaataacc tctgaaagag
     5521 gaacttggtt aggtaccttc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     5581 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     5641 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     5701 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     5761 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     5821 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     5881 gcctaggctt ttgcaaaaag cttgattctt ctgacacaac agtctcgaac ttaaggctag
     5941 agccaccatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     6001 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     6061 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     6121 tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     6181 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     6241 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     6301 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     6361 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     6421 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat
     6481 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     6541 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     6601 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     6661 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     6721 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     6781 cccaacctgc catcacgatg gccgcaataa aatatcttta ttttcattac atctgtgtgt
     6841 tggttttttg tgtgaatcga tagcgataag gatccgcgta tggtgcactc tcagtacaat
     6901 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     6961 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     7021 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     7081 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     7141 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     7201 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     7261 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     7321 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     7381 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     7441 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     7501 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     7561 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     7621 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     7681 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     7741 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     7801 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     7861 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     7921 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     7981 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     8041 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     8101 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     8161 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     8221 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     8281 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     8341 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     8401 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     8461 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     8521 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     8581 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     8641 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     8701 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     8761 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     8821 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     8881 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     8941 catggctcga cagatc
//
