LOCUS       sequence-291310-        7342 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsAGO4-P582GG612FK662AL696Y.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7342)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 7342)
  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-HsAGO4-P582GG612FK662AL696Y_AH
FEATURES             Location/Qualifiers
     source          1..7342
                     /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    4078..4199
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4115..4134)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4169..4188
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4206..4661)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4343..4364)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4555..4574
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4688..4792
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4794..5151
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4794..4985
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4817..4837)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5002..5137
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5186..5980
                     /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             5186..5980
                     /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(5240..5259)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5850..5869
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6168..6187)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6212..6259
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6588..7176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7077..7096
                     /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 caagcttcga attctgcagt cgacatggag
     1381 gcgctgggac ccggacctcc ggctagcctg tttcagccac ctcgtcgtcc tggccttgga
     1441 actgttggaa aaccaattcg actgttagcc aatcattttc aggttcagat tcctaaaata
     1501 gatgtgtatc actatgatgt ggatattaag cctgaaaaac ggcctcgtag agtcaacagg
     1561 gaggtagtag atacaatggt gcggcacttc aagatgcaaa tatttggtga tcggcagcct
     1621 gggtatgatg gcaaaagaaa catgtacaca gcacatccac taccaattgg acgggatagg
     1681 gttgatatgg aggtgactct tccaggcgag ggtaaagacc aaacatttaa agtgtctgtt
     1741 cagtgggtgt cagttgtgag ccttcagttg cttttagaag ctttggctgg gcacttgaat
     1801 gaagtcccag atgactcagt acaagcactt gatgttatca caagacacct tccctccatg
     1861 aggtacaccc cagtgggccg ttcctttttc tcacccccgg aaggttacta ccaccctctg
     1921 ggagggggca gggaggtctg gtttggtttt catcagtctg tgagacctgc catgtggaat
     1981 atgatgctca acattgatgt atctgcaact gctttctacc gggctcagcc tatcattgag
     2041 ttcatgtgtg aggttttaga cattcagaac atcaatgaac agaccaaacc tctaacagac
     2101 tcccagcgtg tcaaatttac caaagaaatc agaggtctca aagttgaggt gacccactgt
     2161 ggacagatga aacgaaaata ccgagtttgt aatgtgacta gacggccagc cagtcatcaa
     2221 acttttcctt tgcagctaga aaacggtcaa gctatggaat gtacagtagc tcaatatttt
     2281 aagcaaaagt atagtctgca actgaaatac ccccatcttc cctgtctcca agtgggacaa
     2341 gaacaaaagc atacatactt gccactcgag gtctgtaata tagtggcagg acagcgatgt
     2401 atcaagaagc tcacagacaa tcagacttcc acaatgatca aagctacagc aagatctgct
     2461 cctgacagac aggaagagat cagtagactg gtgaagagca acagtatggt gggtggacct
     2521 gatccatacc ttaaagaatt tggtattgtt gtccacaatg aaatgacaga gctcacaggc
     2581 agggtacttc cagcaccaat gctgcaatat ggaggccgga ataaaacagt agccacaccc
     2641 aaccagggtg tctgggacat gcgaggaaag cagttttatg ctggcattga aattaaagtt
     2701 tgggcagttg cttgttttgc acctcagaaa caatgtaggg aagatttact aaagagtttc
     2761 actgaccagc tgcgtaaaat ctctaaggat gcaggaatgc ccatccaggg tcagccatgt
     2821 ttctgcaagt atgcacaagg tgcagacagt gtggagccta tgtttaaaca tctgaaaatg
     2881 acttatgtgg gcctacagct aatagtggtt atcctgcctg gaaagacacc agtatatgcg
     2941 gaggtgaaac gtgttggaga tacccttcta ggtatggcca cacagtgtgt ccaggtaaaa
     3001 aatgtagtga agacctcacc tcaaaccctt tccaatcttt gcctgaagat aaatgcaaaa
     3061 cttggaggaa ttaacaatgt gcttgtgcct catcaaaggc cctcggtgtt ccagcagggt
     3121 gtcatcttcc tgggagcgga tgtcacacac cccccagcag gggatgggaa gaaaccttcc
     3181 attgctgctg tggttggcag tatggatttc caccccagcc ggtactgtgc caccgttcgg
     3241 gtgcagactt cccggcagga gatctcccaa gagctcctct acagtcaaga ggtcatccag
     3301 gacctgacta acatggttcg agagctgctg attcagttct acaaatccac acgcttcgca
     3361 cccactcgga tcatctatta ccgtggaggg gtatctgagg gacaaatgaa acaggtagct
     3421 tggccagaac taatagcaat tcgaaaggca tgtattagct acgaagaaga ttaccggcca
     3481 ggaataactt atattgtggt gcaaaaaaga catcacacac gactcttctg tgcagataaa
     3541 acagaaaggg tagggaaaag tggcaatgta ccagcaggca ctacagtgga tagtaccatc
     3601 acacatccat ctgagtttga cttttacctc tgtagtcatg caggaattca gggaaccagc
     3661 cgtccctcac attaccaggt cttgtgggat gacaactgct tcactgcaga tgaactccag
     3721 ctactgactt accagctgtg tcacacctat gtgaggtgca ctcgctcagt ctctattcca
     3781 gcccctgcat attatgcccg gcttgtagca tttagggcaa ggtatcatct ggtggataaa
     3841 gatcatgaca gtgcggaagg cagtcatgtg tcaggacaga gcaacggccg ggatcctcag
     3901 gccttggcta aggctgtgca aatccaccat gatacccagc acacgatgta ttttgcctga
     3961 tctagataac tgatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     4021 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     4081 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4141 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     4201 cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4261 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4321 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4381 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4441 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4501 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4561 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4621 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     4681 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4741 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     4801 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4861 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4921 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4981 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5041 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5101 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     5161 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     5221 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     5281 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     5341 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     5401 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5461 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5521 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5581 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5641 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5701 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5761 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5821 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5881 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5941 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     6001 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     6061 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     6121 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     6181 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     6241 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     6301 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     6361 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     6421 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     6481 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     6541 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6601 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6661 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6721 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6781 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6841 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6901 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6961 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     7021 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     7081 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     7141 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     7201 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     7261 tctgtggata accgtattac cgccatgcat tagttattaa tagtaatcaa ttacggggtc
     7321 attagttcat agcccatata tg
//
