LOCUS       sequence-156128-        7885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for expression of HA-tagged Nematostella vectensis AGO1 in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7885)
  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 7885)
  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-1547
FEATURES             Location/Qualifiers
     source          1..7885
                     /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"
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 ggcttctaat aatccagtga
     5341 atcgtgttgg tggtaatttc aaccctccaa aaaggccaaa tcatggctca acaggacgca
     5401 ccattgcact tcgagcaaat ttcttccctg tccagttacc tcgtggtaac attcatcact
     5461 atgacctgtc catatgtcca gacaaatgtc ctcggcgagt caatagggac gttgtagagg
     5521 tcatggtgtt aaattaccac aaagtatttg gtggaatgaa acctgtgttt gatgggagga
     5581 agaatctata cacaagggat cctctaccta ttgggaaaac accgacagag tttgaagttg
     5641 tgctaccgac agacaacagc caagataaga aattcaaggt gacattgaag tgggtgtcac
     5701 aagtaagttt gtatgctcta gagaaggcct tggagggcac atgtaaccag ataccatttg
     5761 agaccatcca ggctcttgat gtggtactac ggcatctgcc atccatgaag tacacgcctg
     5821 ttggcagatc cttcttctcc cccccagatg gctatgactt cccactcgaa agagggaggg
     5881 aggtttggtt cggctttcat caaagtatcc gaccctctca gtggaagatg ctactgaaca
     5941 ttgatgtatc tgcaacagca ttttacaagt gccagtctgt ggttgaattc atgtgtgaag
     6001 ttcttcgcat aaggaaagat gatctggaga aacatgcacc tctcaacgat gcagatagac
     6061 ttcgctttac cagagaaata aaaggactta aagttgaaat tactcactgt gggccaatga
     6121 aacgcaagta cagggtcatc aatgtgacca agcaaccagc ccaggccttg cagttccccc
     6181 ttacactaga gtctggtcag acagcacaga taactgtcgc tcgctacttc caggacaagc
     6241 ataagcaaag actcagatat cctcaccttc cttgtcttca agttggccaa gagcagcgcc
     6301 acacctatct tcctctcgag gtgtgtaaca tagtgcctgg ccagcgctgc gtcaagaagc
     6361 ttacagacac ccagacatct aagatgatcc gagcgacagc acggtctgca ccagataggg
     6421 agagggaaat ccgaggactg gtcaagaagg ccaactttga tgaggacgcg tacgtaaagg
     6481 actttagtat ttccatcggc aaaaacatgg ttgagcttca aggcagagtt ctacctcctc
     6541 caaaacttgt ctacggagga aagcaaagct cacctatcac tccaaaagga ggggtatggg
     6601 acatgagagg taggcagctt ttccacggca ttgagatcag gacgtgggcc attgcttgtt
     6661 ttgtcaagca acagatgtgt acagaggact ctctacgaag gttctccaat cagctgatga
     6721 agatcagtgt agagcaaggc atgcctatct cctgcccgcc tgtcttcttc cgctatgcgc
     6781 gcaacccaga cgaggtcgag cgaatgttcc gccggctgaa agaggcacac cccgacctgc
     6841 aaatgatcct tgttgtttta ccgggaaaga cgcctgtgta tgccgaggtg aagcgcgtcg
     6901 gtgacacaat gctaggcgtg ataacgcaat gcatccaggg gaaaaatgtc actaaaccct
     6961 caccccagac cctctccaat ctctgtctca agataaacgc caagcttggc ggcgtcaaca
     7021 atatactcgc cccagaaata cgaccccctg tgttccggga gccggtgata ttccttggcg
     7081 ctgatgtgac acatcccgct gctggtgacg ataagcgacc atctgtagct gctgtggtgg
     7141 ggagcatgga tgcgcacccc agtcgctact atgcctctgt gcgggtgcag actcacagac
     7201 aggaaatcat cgcggagctg gcagccatgg tgcgagagct tcttgtgcag ttctaccgct
     7261 cgacaaggca caaaccgcaa aggatagtgt tttaccgaga tggagtcagc gagggtcaat
     7321 tccgacaggt tcttgtgcac gagttgaagg cgatacgcga ggcgtgtatc aagcttgagg
     7381 tgggatatca acccggaatc tctttcatcg tggtacagaa gcgtcaccac acccgcctct
     7441 tctgccaaag agaccaagac aagtgtggta agagtggcaa tgtgcccccc ggcaccaccg
     7501 tggaccgagg gatcacgcac ccaaccgaat ttgacttcta cctgtgcagc cacgccggca
     7561 tacagggtac gagtagaccg tcgcattacc acgtgctgtg ggacgataat aacttctccg
     7621 cagacgagct ccaggccctc acctaccaac tctgccacac atacgtgaga tgcacgcgag
     7681 ccgtgtctat accagcacct gcctactacg cgcatttggt cgcgttccgc gcgcgatacc
     7741 atatgatgga tagggacaag gaagcgggaa atgataacag ttccgttgcc agcggtgaag
     7801 attgcagggg acctcaacta ctcgcaaacg ccgtgcaagt tcaccccaag gtcctacagg
     7861 gaatgtactt tgcttgaagg cggcc
//
