LOCUS       sequence-291308-        7318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of HsAGO1-P588GA618FK658AL692Y.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7318)
  TITLE     Izaurralde lab plasmids
REFERENCE   2  (bases 1 to 7318)
  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-HsAGO1-P588GA618FK658AL692Y_AH
FEATURES             Location/Qualifiers
     source          1..7318
                     /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    4054..4175
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4091..4110)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4145..4164
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4182..4637)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4319..4340)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4531..4550
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4664..4768
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4770..5127
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4770..4961
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4793..4813)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4978..5113
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5162..5956
                     /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             5162..5956
                     /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(5216..5235)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5826..5845
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6144..6163)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6188..6235
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6564..7152
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7053..7072
                     /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 atggaagcgg gaccctcggg agcagctgcg
     1381 ggcgcttacc tgccccccct gcagcaggtg ttccaggcac ctcgccggcc tggcattggc
     1441 actgtgggga aaccaatcaa gctcctggcc aattactttg aggtggacat ccctaagatc
     1501 gacgtgtacc actacgaggt ggacatcaag ccggataagt gtccccgtag agtcaaccgg
     1561 gaagtggtgg aatacatggt ccagcatttc aagcctcaga tctttggtga tcgcaagcct
     1621 gtgtatgatg gaaagaagaa catttacact gtcacagcac tgcccattgg caacgaacgg
     1681 gtcgactttg aggtgacaat ccctggggaa gggaaggatc gaatctttaa ggtctccatc
     1741 aagtggctag ccattgtgag ctggcgaatg ctgcatgagg ccctggtcag cggccagatc
     1801 cctgttccct tggagtctgt gcaagccctg gatgtggcca tgaggcacct ggcatccatg
     1861 aggtacaccc ctgtgggccg ctccttcttc tcaccgcctg agggctacta ccacccgctg
     1921 gggggtgggc gcgaggtctg gttcggcttt caccagtctg tgcgccctgc catgtggaag
     1981 atgatgctca acattgatgt ctcagccact gccttttata aggcacagcc agtgattgag
     2041 ttcatgtgtg aggtgctgga catcaggaac atagatgagc agcccaagcc cctcacggac
     2101 tctcagcgcg ttcgcttcac caaggagatc aagggcctga aggtggaagt cacccactgt
     2161 ggacagatga agaggaagta ccgcgtgtgt aatgttaccc gtcgccctgc tagccatcag
     2221 acattcccct tacagctgga gagtggacag actgtggagt gcacagtggc acagtatttc
     2281 aagcagaaat ataaccttca gctcaagtat ccccatctgc cctgcctaca agttggccag
     2341 gaacaaaagc atacctacct tcccctagag gtctgtaaca ttgtggctgg gcagcgctgt
     2401 attaaaaagc tgaccgacaa ccagacctcg accatgataa aggccacagc tagatccgct
     2461 ccagacagac aggaggagat cagtcgcctg atgaagaatg ccagctacaa cttagatccc
     2521 tacatccagg aatttgggat caaagtgaag gatgacatga cggaggtgac agggcgagtg
     2581 ctgccggcgc ccatcttgca gtacggcggc cggaaccggg ccattgccac acccaatcag
     2641 ggtgtctggg acatgcgggg gaaacagttc tacaatggga ttgagatcaa agtctgggcc
     2701 atcgcctgct tcgcacccca aaaacagtgt cgagaagagg tgctcaagaa cttcacagac
     2761 cagctgcgga agatttccaa ggatgcgggg atgcctatcc agggtcaacc ttgtttctgc
     2821 aaatatgcac agggggcaga cagcgtggag cctatgttcc ggcatctcaa gaacacctac
     2881 tcagggctgc agctcattat tgtcatcctg ccagggaaga cgccggtgta tgctgaggtg
     2941 aaacgtgtcg gagatacact cttgggaatg gctacgcagt gtgtgcaggt gaagaacgtg
     3001 gtcaagacct cacctcagac tctgtccaac ctctgcctca agatcaatgt caaacttggt
     3061 ggcattaaca acatcctagt cccacaccag cgctctgccg tttttcaaca gggagtgata
     3121 ttcctgggag cagatgttac acacccccca gcaggggatg ggaaaaaacc ttctatcaca
     3181 gcagtggtag gcagtatgga ttttcacccc agccgatact gtgctactgt gcgggtacag
     3241 cgaccacggc aagagatcat tgaagacttg tcctacatgg tgcgtgagct cctcatccaa
     3301 ttctacaagt ccacccgttt cgcgcctacc cgcatcatct tctaccgaga tggggtgcct
     3361 gaaggccagc taccccagat actccactat gagctactgg ccattcgtga tgcctgcatc
     3421 aaatacgaaa aggactacca gcctgggatc acttatattg tggtgcagaa acgccatcac
     3481 acccgccttt tctgtgctga caagaatgag cgaattggga agagtggtaa catcccagct
     3541 gggaccacag tggacaccaa catcacccac ccatttgagt ttgacttcta tctgtgcagc
     3601 cacgcaggca tccagggcac cagccgacca tcccattact atgttctttg ggatgacaac
     3661 cgtttcacag cagatgagct ccagatcctg acgtaccagc tgtgccacac ttacgtacga
     3721 tgcacacgct ctgtctctat cccagcacct gcctactatg cccgcctggt ggctttccgg
     3781 gcacgatacc acctggtgga caaggagcat gacagtggag aggggagcca catatcgggg
     3841 cagagcaatg ggcgggaccc ccaggccctg gccaaagccg tgcaggttca ccaggatact
     3901 ctgcgcacca tgtacttcgc ttgaggatcc accggatcta gataactgat cataatcagc
     3961 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     4021 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     4081 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4141 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     4201 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     4261 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     4321 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     4381 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     4441 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     4501 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     4561 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     4621 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4681 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4741 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     4801 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4861 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4921 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4981 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     5041 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     5101 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     5161 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     5221 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     5281 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     5341 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     5401 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     5461 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     5521 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     5581 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     5641 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     5701 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5761 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5821 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5881 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5941 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     6001 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     6061 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     6121 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     6181 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     6241 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     6301 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     6361 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     6421 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     6481 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6541 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6601 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6661 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6721 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6781 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6841 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6901 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6961 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7021 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7081 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7141 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7201 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     7261 atgcattagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatg
//
