LOCUS       sequence-156246-        9453 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of GST-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 9453)
  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 9453)
  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-1479
FEATURES             Location/Qualifiers
     source          1..9453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(514..531)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(514..530)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(523..545)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(798..1178)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(860..881)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1072..1091
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(1215..1234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1334..1356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1394..1412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1480..1584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1585..1653,1654..2445)
                     /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(1803..1822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2616..3204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3105..3124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3358..3375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3492..3513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3528..3533,3534..3551,3552..3558)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3566..3582
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3571..3593
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3590..3606
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3590..3606
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3635..3845
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3657..3853
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3704..3839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3942..5120
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4173..5111
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5068..5088
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5172..5825
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     5783..5805
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             5832..5849
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
ORIGIN
        1 ccttgcaccc ctcactctgg cctcagccga ctgcacagcg gcccctgcat ccccttggct
       61 gtgaggcccc tggacaagca gaggtggcca gagctgggag gcatggccct ggggtcccac
      121 gaatttgctg gggaatctcg tttttcttct taagactttt gggacatggt ttgactcccg
      181 aacatcaccg acgcgtctcc tgtttttctg ggtggcctcg ggacacctgc cctgccccca
      241 cgagggtcag gactgtgact ctttttaggg ccaggcaggt gcctggacat ttgccttgct
      301 ggacggggac tggggatgtg ggagggagca gacaggagga atcatgtcag gcctgtgtgt
      361 gaaaggaagc tccactgtca ccctccacct cttcaccccc cactcaccag tgtcccctcc
      421 actgtcacat tgtaactgaa cttcaggata ataaagtgct tgcctccaaa aaaaaaaaaa
      481 aaaaaaaaaa aaaaaaaaaa aaaaaaagaa ttcactggcc gtcgttttac aacgtcgtga
      541 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
      601 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
      661 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      721 caaaacgtta atattttgtt aaaattcggt ccacgctggt ttgccccagc aggcgaaaat
      781 cctgtttgat ggtggttccg aaatcggcaa aatcccttat aaatcaaaag aatagcccga
      841 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      901 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      961 caaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     1021 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     1081 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     1141 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtac tatggttgct ttgacgagca
     1201 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     1261 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     1321 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     1381 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     1441 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1501 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     1561 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     1621 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     1681 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     1741 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     1801 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     1861 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     1921 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1981 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     2041 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     2101 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     2161 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     2221 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     2281 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     2341 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     2401 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     2461 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     2521 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     2581 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2641 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2701 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2761 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2821 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2881 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2941 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3001 cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3061 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3121 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3181 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3241 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3301 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3361 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     3421 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     3481 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     3541 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     3601 tatgaccatg attacgccaa gcttaggctc cccagcaggc agaagtatgc aaagcatgca
     3661 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3721 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3781 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3841 aggcttttgc aaaaagcttt gcaaagatgg ataaagtttt aaacagagag gaatctttgc
     3901 agctaatgga ccttctaggt cttgaaagga gtgcctcgtg aggctccggt gcccgtcagt
     3961 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
     4021 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
     4081 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
     4141 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
     4201 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
     4261 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
     4321 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
     4381 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
     4441 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     4501 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     4561 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     4621 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     4681 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     4741 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     4801 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     4861 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     4921 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     4981 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     5041 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     5101 ttccatttca ggtgtcgtga ggaattctct agagatccct cgacctcgag atccattgtg
     5161 ctggatctac aatggcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
     5221 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
     5281 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
     5341 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
     5401 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
     5461 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     5521 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     5581 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     5641 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     5701 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     5761 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     5821 caaaatcgga tctggttccg cgtggatctc ctgcaggcat gcctaaggat gacaagaagg
     5881 aattcctcaa gtcattgggc gttgacaccc tgtcctctga aaacgacatc aagagagcct
     5941 ataagaaaat agcactccag tgtcaccctg ataagaaccc cagcgacacg tccgccagaa
     6001 aacaattcga ggaggtctct aaagcattcg agtacctcgt ccacgacggg atcaatgaca
     6061 gcctgaaaga ttacgataag gttttctttg atttcctccg ggacatattg ttcggcagcg
     6121 atggtgcttc agacttcttt acctattttt gtggttccaa gcagcacagc tgtatctcca
     6181 cagacaccga ccctgatttt agatatcagg atgacgacga ggaggaggac attgatgggg
     6241 aacaggagct ggaaagcctc ctggagatcg tcaagaaccg ccggaagcgc gggaagcccg
     6301 tacccgatga gtttcacgag gagaaactgc aggagctgaa gactcgactg tctcagcaca
     6361 aagaggacat gaaaaaggat gggagcaaga aacaggctgt taagaagggg aaagctgtca
     6421 gcgtccctgt ggcaaaaccc aagccaaaga gcaaaaagca gctccttgct gaacaacggc
     6481 ggcgggctaa agaaatggag cagatagcca tggaactgga ggagaaaaga aaagaagaag
     6541 aagaaaagga gaagcaacgc aaacagctgg aagtagagaa acagaaacgc cttgaggagg
     6601 agcgcatcag gtcacagaac gaggagcgaa aacggcggga gcgagagcaa aaagaacggg
     6661 agaagcagca gaaactggat atggagaaag aggagcgccg ccggcgccag caggaggagg
     6721 agaagaaaag acgagaggag gaagagaaga gaaaaaaagt cgagaaagag aagcaggaaa
     6781 gggagaaggc cgcaaaaaaa gctgccctcc aggctgcgaa ggagaaagaa gaacaggcga
     6841 aggctgcatc taaaaatcag aatattaaaa gacagcagca acaacagcga actgatcaac
     6901 agcagtatcc tagggaaata cctcctaggt ttcagaaaat gaaaagacag caacaacagc
     6961 agcagctgca gactccaaac ttgccacctc cagccccaac cggctctccg gctcagctta
     7021 acaagttcca gtcaaaacct cagcaacagc ctgttggcaa gaagaaggat cctaaccgct
     7081 tcgctagcaa ggagtacgct gacctcgttg acgaggtgga cgatgcgggc tggaacaagg
     7141 gatctcagtg taaaggctct ggatgggaag ggactagcga ggattgggag gagaacatcg
     7201 ccgaaaagtg gggcgcagat aggcagtggg ggagcaagga agggtccagt aaccaactcg
     7261 ttgtggaccg cacagatggc gatagctggc ccaccgtggg cagtaagtct gctattggag
     7321 ataagcccaa ttcttccctt gagaacagta acagcaaaaa cagcagtgag accgtgcctg
     7381 tggggacaat cggcagcaaa cctccaagca gcaattggaa cagtaacagg aacagcggag
     7441 gactcccact gtccgagaat tgggatacaa cctgtgacga gagcatcgat cgcgctctgg
     7501 acgccactga atctaaagga tcagtggggc ctgggggagt caattgggtt ggcatgggta
     7561 caggcggatt gggcgggatc attggaaccc ctttgggttg ggagcctagc aaagagaacc
     7621 gcgatattga taagaccgcc tctaaaagcg gctggtgcgg ggaacagaac tctactgtcg
     7681 atagctggaa taagactcca ggtaagggcc cgaaaacaca ggtgttcggt ggatcagccc
     7741 ccacatgggg gggaacaccg gcagactctt cttccgcctg ggtggagact agcgggactg
     7801 agaccaataa acccaactcc tccggctgga atagcaaggt cagcagcggc ctgagtcagg
     7861 gaaactctag tagcatggcc tctgtagata gtaccgacaa taaaccaaca gacaataaac
     7921 cagctccatc aggctggggc gaagccggcc catcatgggc tgtgcaggct tccgttaccg
     7981 gaacagcatg ctgggacacc gccactacca catcccctaa aaagcctctg gattccaaag
     8041 atagcaagct gagccacgat aaggtgtccg ggtggctcga gacatccgtg acctccacta
     8101 gcgtgactgc agattggaac ggcgatagca atgctgacca ggaggactac tacgaagagg
     8161 cgtgggacgg gtggacaact gcatcctcca agcgaaacaa ggccccgaag ccggcaccca
     8221 caaatgactc caatgcatgg atgtccagga gccttaaaca gcttctggac atgggattta
     8281 aacaagagga cgctgagaaa gcgctgcgcg ccaatcacat gaacatcgag agcgcgattt
     8341 ctgacctgct gagccttaca gcctcccaga tcgaggaagc ctcccgagct agttctgcta
     8401 acaccggtga gcctgacccg gcagaagcct gtaggatgca gcagcagaat actactacat
     8461 ctaagccaaa ccgaaaacaa aggcgcaaga cccagcagca gttgcagcaa cagcaggcct
     8521 ctcaggccaa gcaggagtcc aagcaaaccg atatctacga tgctaatagt cagtcagggt
     8581 cttctgactc tacagttaag gatcccaaat ctgagtccaa taaagataaa gaaggccagg
     8641 ccgccgaggg ccagagtaat aagccttcta atcagggggc tagcgacggg aaagggaacg
     8701 cctcaaaaac cacagaaaat tctccacacc tctcaccggg taagtccaat gcaggtcaga
     8761 aacgagccac aactggcctc atccagacgc ctgcattggg gaccccgacc ccaatgagaa
     8821 ctccgatgac acatcctttg ttgcaccagc agtttctgca tcaggtgcag ctccaccagc
     8881 tgaggatggc ccagggaggt ctccttcaac ctcccataaa cccccagcaa gtagctttgt
     8941 tgcagcagat ggctcagctt cagatggtcc agcatcgcat cgcccagcag caactgaccc
     9001 agaagcacaa catccaggca caacagcagc tgtatcaaca gcagcaacag attgcactca
     9061 tgatggggca gctgcagcag caagtgatgc agcagcacaa tcatcagata gccggttccc
     9121 ggttccccag ccccggcttt cagcagagca cccttcctcc tcaaagcagt tccgcccagc
     9181 ctggtactcc acctcagaac gccaagaatt ccaccaaagc cacttctact gagcctagtc
     9241 aatcaaaacc tgaggaaccc cccaaggtgt ccaccgcaga taagcctgtg accacagttg
     9301 ccagcggaga gcgaataact cagcagagcc cttgagcggc cgcgactcta gagtgagggt
     9361 ccccacctgg gacccttgag agtatcaggt ctcccacgtg ggagacaaga aatccctgtt
     9421 taatatttaa acagcagtgt tccccatctg ggt
//
