LOCUS       sequence-156127-        8620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of HA-tagged Nematostella vectensis AGO2 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8620)
  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 8620)
  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-1548
FEATURES             Location/Qualifiers
     source          1..8620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(626..643)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(626..642)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(635..657)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(910..1290)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(972..993)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1184..1203
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(1327..1346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1446..1468
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1506..1524)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1592..1696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1697..1765,1766..2557)
                     /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(1915..1934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2728..3316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3217..3236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3470..3487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3604..3625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3640..3645,3646..3663,3664..3670)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3678..3694
                     /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     3683..3705
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3702..3718
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3702..3718
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3747..3957
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3769..3965
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3816..3951
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4054..5232
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4285..5223
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5180..5200
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5283..5309
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7227..7238
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 gcgactctag agtgagggtc cccacctggg acccttgaga gtatcaggtc tcccacgtgg
       61 gagacaagaa atccctgttt aatatttaaa cagcagtgtt ccccatctgg gtccttgcac
      121 ccctcactct ggcctcagcc gactgcacag cggcccctgc atccccttgg ctgtgaggcc
      181 cctggacaag cagaggtggc cagagctggg aggcatggcc ctggggtccc acgaatttgc
      241 tggggaatct cgtttttctt cttaagactt ttgggacatg gtttgactcc cgaacatcac
      301 cgacgcgtct cctgtttttc tgggtggcct cgggacacct gccctgcccc cacgagggtc
      361 aggactgtga ctctttttag ggccaggcag gtgcctggac atttgccttg ctggacgggg
      421 actggggatg tgggagggag cagacaggag gaatcatgtc aggcctgtgt gtgaaaggaa
      481 gctccactgt caccctccac ctcttcaccc cccactcacc agtgtcccct ccactgtcac
      541 attgtaactg aacttcagga taataaagtg cttgcctcca aaaaaaaaaa aaaaaaaaaa
      601 aaaaaaaaaa aaaaaaaaag aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
      661 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
      721 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
      781 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaaaacgt
      841 taatattttg ttaaaattcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
      901 atggtggttc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagggt
      961 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     1021 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa
     1081 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     1141 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     1201 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     1261 ccgcgcttaa tgcgccgcta cagggcgcgt actatggttg ctttgacgag catggtgcac
     1321 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     1381 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     1441 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     1501 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     1561 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1621 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2581 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2641 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2701 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2761 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2821 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     2881 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2941 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3001 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3061 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca
     3121 ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3181 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3241 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3301 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3361 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3421 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3481 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3541 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3601 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     3661 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     3721 tgattacgcc aagcttaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3781 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3841 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3901 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3961 gcaaaaagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt gcagctaatg
     4021 gaccttctag gtcttgaaag gagtgcctcg tgaggctccg gtgcccgtca gtgggcagag
     4081 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc
     4141 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     4201 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     4261 aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc
     4321 tttacgggtt atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt
     4381 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga
     4441 gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga
     4501 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat
     4561 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa
     4621 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
     4681 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
     4741 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
     4801 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
     4861 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
     4921 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
     4981 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg
     5041 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag
     5101 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag
     5161 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt
     5221 caggtgtcgt gaggaattct ctagagatcc ctcgacctcg agatccattg tgctggatgg
     5281 cttacccata cgatgttcca gattacgctc ctgcaggcat gccgaagaaa tcgaaaggac
     5341 gtggaagagg gagaggaaga ggaaaccctc atcatgaaaa acaacaaact ctagttgggc
     5401 aacaagcgac ttcccgaaat aacgagcata aacaactccc caagccaaca cagacccaac
     5461 aagaaacatc attatcccag cagccatata cctgtagcag tgcagcagaa gcgcaaggac
     5521 ccctgacccc gcccaataac actcaagggg ggagtcttac caatgcagaa ccggcacagg
     5581 ctgatacatt ggggcaaaaa tttgaaactc aactaaattt aagcccacaa agtggcaaag
     5641 agcagggtgc gatccctaaa acaggagcta gacttaaggg gaacagcttg gcaccaggct
     5701 cacagggcaa tggacaattt tcctctaaga acctgctggc tcagatgcag agaacaccaa
     5761 gaagtgcatc agagtcccag aacgaagctt catccaaatc acagcaagca cacaacaatc
     5821 aaagccaggc tgcagcccat cagcagactc aagcaggtcc acagcagact ccagcacgtt
     5881 cacagcagac tccagcaggt ccacagcaga ctcaagcagg tccacagcag actccagcag
     5941 gtccacagca gactcaagca ggtccacagc agactcaaga aggtccacag cagactccag
     6001 cacgttcaca gcagacccca gcaataatag ggtcaacaac aactgctgat gaacacctgg
     6061 ctcgtcacag gcaggaaaac atggaattac cgaagcggcc aggttatgga agggagggaa
     6121 ggaaaataaa actgcgagct aacttcttcc aagtgactct tccaaacgtt gatttcctat
     6181 atcactacga tctggagatt tctcctgaga aagccccagt gtctgtttgt cgtgatgtgg
     6241 tggacgctgc tataaagcat ggggagttca agggagtttt caatggatgc aaacctgctt
     6301 ttgatgggag gagaaatttg tactgcaggg agccattgcc tttaaagtca gaggaagctt
     6361 ccctgaaagt cactcttcct ggaactgatg ggggaaaaga acgaaagttt acactcaaaa
     6421 ttaaggaagc aggtctggtt agcatcaaag agcttgacca gttcctaaat ggggagttcc
     6481 gtggaaaggt tccccaagac gctatccagg gtatggatat tgtcctgaga cagatgccgt
     6541 ccatgaagtt cacagctgtg gggaggtgct tcttcccacc tcccaacggc cactgccatg
     6601 atcttggggg tggctgtgaa ttatggacgg ggttctatca aagtgtgcgg ccgtctcagt
     6661 ggaaaaccat gctgctgaac atcgatgtat cctctaaagg cttccagaaa tcgatgccgg
     6721 tgatagactt catgttggag attcttagac aggatcggaa cagagttctt gacacacgtt
     6781 gggtgatgga cgaaagagac aagaagaagc tcaccacgga gataaaaggt cttcgcgtag
     6841 agactaccca cattaaaaga aagttcactg tgatgggtct gtcacaccct gcatttaaca
     6901 accgctttag gcttgaagac aacaccgaga ccaccgtaga ggcgtacttc cgcaacaagt
     6961 acaatatatc tctcaggtac ccccatttac cgtgcctact ggttggtcaa aagaagaact
     7021 ccgtgccaat ggaggtgtgt aacatgattc ccactcagcg gaagcgtctg acggatgaac
     7081 aaactgcggc gatgattcgc aaaacagcaa agccagccaa tgaacgccag cgggacatca
     7141 accaatgggt cgatgagttg gcaacagcaa gtgaccagta cctgaaaaac gaatacggca
     7201 tgcgaatcaa caagcagatg gtagcaatcg aagggcgtgt cctacccgca cccgagctta
     7261 ccctcggggg caatccgcag ggtagcgccc ttaccccctc tgacggagcg tgggacatga
     7321 ggggaaagtc gtttttcgag gcaaggactg ttgaggtctg ggcactggtg tgcttttcac
     7381 atccaaagtg gtgtcctaag gagaagctgg aggggtttgc tcggcaaatg ggcaatgttt
     7441 gtcgaagtga aggaatgaga atgaacccgg taccttgtcg cgcggagtat gcgtcacgcg
     7501 tgcaagaggt tgagggaata tttggcaagc ttcttcacga ctttaatagt ctgcaattga
     7561 ttgtggtcgc tctcccggac aggggcaaca aagatgtcta taacgaggtg aagagagtcg
     7621 gcgacacagt cttaggtatt cccacccagt gtgttcaaat gaagcaattt acgatggcta
     7681 aacctcaagt gtgctcgaac atcgccatga agatcaatgg caaactaggc ggcaccaacc
     7741 acgtgatcgc cgatagcctc aaggcgacaa tcactgatga caaaggcaat ggaatattta
     7801 actccccggt aatcatattt ggcgctgatg tcacccaccc agcccctggt gacaatggca
     7861 tcccttctat cgccgccgtc gtcgccagcc tgaatcgcaa cgctagccgc tactgcgcac
     7921 gcgtgagacc gcagacccac atgaagtgca agcaggctca ggagatcatt gttgatctgg
     7981 cagacatggt gaaagaactc atgattgagt tttataagga gaacaaaagg aagccggtgc
     8041 ggattatctt ttatcgggat ggtgtcagtg agggtcagtt tcagcaggtt ctcctcgagg
     8101 aggtccgtgc tgttcagcag gcgtgcgcga tgctcgagaa agactatcag cctctcatca
     8161 cgtttgtggt cgtgcagaaa agacaccacg cgcgactttt tgccgaggaa ggcagggacg
     8221 cgagggggaa atcgagaaat gtcccagctg gcacgacggt ggacacggtt atatgtcacc
     8281 cgttcgagtt tgacttctat ctctgcagtc atgctggtat tcagggtacg agccggccca
     8341 cccattacca cgtgctgtac gatgataacg ggttctcggc agataagctg caggcgctca
     8401 cctatcaact ctgccatacg ttcgcaagat gcacacgcag cgtgtctatg cctgcaccgg
     8461 cctactacgc acaccttgcc gcgtttagag ccagggggct tgtcaacgca ggacgcggcg
     8521 cagcaaatgg gtctgaggag agtcaaatga aaacctacta cgagacaatc aaagtgtgca
     8581 agcgaatgga agcatccatg tactttacct aaaggcggcc
//
