LOCUS       sequence-291309-        7327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsAGO3-P591GA621FK661AL695Y.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7327)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 7327)
  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"
COMMENT     Sequence Label: pT7-EGFP-C1-HsAGO3-P591GA621FK661AL695Y_AH
FEATURES             Location/Qualifiers
     source          1..7327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        9..312
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        313..516
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     467..487
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     561..580
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        561..579
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4063..4184
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4100..4119)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4154..4173
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4191..4646)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4328..4349)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4540..4559
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4673..4777
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4779..5136
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4779..4970
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4802..4822)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4987..5122
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5171..5965
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5171..5965
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5225..5244)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5835..5854
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6153..6172)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6197..6244
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6573..7161
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7062..7081
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
       61 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      121 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      181 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      241 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      301 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      361 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      421 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      481 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      541 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc tctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct atggaaatcg gctccgcagg acccgctggg
     1381 gcccagcccc tactcatggt gcccagaaga cctggctatg gcaccatggg caaacccatt
     1441 aaactgctgg ctaactgttt tcaagttgaa atcccaaaga ttgatgtcta cctctatgag
     1501 gtagatatta aaccagacaa gtgtcctagg agagtgaaca gggaggtggt tgactcaatg
     1561 gttcagcatt ttaaagtaac tatatttgga gaccgtagac cagtttatga tggaaaaaga
     1621 agtctttaca ccgccaatcc acttcctgtg gcaactacag gggtagattt agacgttact
     1681 ttacctgggg aaggtggaaa agatcgacct ttcaaggtgt caatcaaatt tgtctctcgg
     1741 gtgagttggc acctactgca tgaagtactg acaggacgga ccttgcctga gccactggaa
     1801 ttagacaagc caatcagcac taaccctgtc catgccgttg atgtggtgct acgacatctg
     1861 ccctccatga aatacacacc tgtggggcgt tcatttttct ccgctccaga aggatatgac
     1921 caccctctgg gagggggcag ggaagtgtgg tttggattcc atcagtctgt tcggcctgcc
     1981 atgtggaaaa tgatgcttaa tatcgatgtt tctgccactg ccttctacaa agcacaacct
     2041 gtaattcagt tcatgtgtga agttcttgat attcataata ttgatgagca accaagacct
     2101 ctgactgatt ctcatcgggt aaaattcacc aaagagataa aaggtttgaa ggttgaagtg
     2161 actcattgtg gaacaatgag acggaaatac cgtgtttgta atgtaacaag gaggcctgcc
     2221 agtcatcaaa cctttccttt acagttagaa aacggccaaa ctgtggagag aacagtagcg
     2281 cagtatttca gagaaaagta tactcttcag ctgaagtacc cgcaccttcc ctgtctgcaa
     2341 gtcgggcagg aacagaaaca cacctacctg ccactagaag tctgtaatat tgtggcaggg
     2401 caacgatgta tcaagaagct aacagacaat cagacttcca ctatgatcaa ggcaacagca
     2461 agatctgcac cagatagaca agaggaaatt agcagattgg taagaagtgc aaattatgaa
     2521 acagatccat ttgttcagga gtttcaattt aaagttcggg atgaaatggc tcatgtaact
     2581 ggacgcgtac ttccagcacc tatgctccag tatggaggac ggaatcggac agtagcaaca
     2641 ccgagccatg gagtatggga catgcgaggg aaacaattcc acacaggagt tgaaatcaaa
     2701 atgtgggcta tcgcttgttt tgccacacag aggcagtgca gagaagaaat attgaagggt
     2761 ttcacagacc agctgcgtaa gatttctaag gatgcaggga tgcccatcca gggccagcca
     2821 tgcttctgca aatatgcaca gggggcagac agcgtagagc ccatgttccg gcatctcaag
     2881 aacacatatt ctggcctaca gcttattatc gtcatcctgc cggggaagac accagtgtat
     2941 gcggaagtga aacgtgtagg agacacactt ttgggtatgg ctacacaatg tgttcaagtc
     3001 aagaatgtaa taaaaacatc tcctcaaact ctgtcaaact tgtgcctaaa gataaatgtt
     3061 aaactcggag ggatcaataa tattcttgta cctcatcaaa gaccttctgt gttccagcaa
     3121 ggagtgatct ttttgggagc cgatgtcact catccacctg ctggtgatgg aaagaagcct
     3181 tctattgctg ctgttgtagg tagtatggat tttcacccaa gcagatactg tgccacagta
     3241 agagttcaga gaccccgaca ggagatcatc caggacttgg cctccatggt ccgggaactt
     3301 cttattcaat tttataagtc aactcggttc gcgcctactc gtatcatctt ttatcgggat
     3361 ggtgtttcag aggggcagtt taggcaggta ttatattatg aactactagc aattcgagaa
     3421 gcctgcatca gttacgagaa agactatcaa cctggaataa cctacattgt agttcagaag
     3481 agacatcaca ctcgattatt ttgtgctgat aggacagaaa gggttggaag aagtggcaat
     3541 atcccagctg gaacaacagt tgatacagac attacacacc catatgagtt cgatttttac
     3601 ctctgtagcc atgctggaat acagggtacc agtcgtcctt cacactatca tgttttatgg
     3661 gatgataact gctttactgc agatgaactt cagctgctaa cttaccagct ctgccacact
     3721 tacgtacgct gtacacgatc tgtttctata cctgcaccag cgtattatgc tcacctggta
     3781 gcatttagag ccagatatca tcttgtggac aaagaacatg acagtgctga aggaagtcac
     3841 gtttcaggac aaagcaatgg gcgagatcca caagctcttg ccaaggctgt acagattcac
     3901 caagatacct tacgcacaat gtacttcgct taaggatcca ccggatctag ataactgatc
     3961 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     4021 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     4081 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4141 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     4201 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     4261 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     4321 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     4381 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     4441 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     4501 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     4561 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     4621 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     4681 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4741 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     4801 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4861 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     4921 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     4981 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     5041 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     5101 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     5161 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     5221 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     5281 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     5341 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     5401 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     5461 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     5521 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     5581 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     5641 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     5701 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     5761 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     5821 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     5881 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5941 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     6001 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     6061 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     6121 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     6181 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     6241 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     6301 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     6361 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     6421 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6481 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6541 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6601 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6661 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     6721 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6781 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6841 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6901 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6961 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7021 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7081 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7141 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7201 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     7261 attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc
     7321 atatatg
//
