LOCUS       sequence-422103-        8521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression plasmid for generating stable FCGR3A 
            (CD16a)-expressing cell lines..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8521)
  AUTHORS   Vincken R, Ruiz-Saenz A
  TITLE     A co-culture model system to quantify antibody-dependent cellular 
            cytotoxicity in human breast cancer cells using an engineered 
            natural killer cell line.
  JOURNAL   STAR Protoc. 2023 Apr 17;4(2):102224. doi: 
            10.1016/j.xpro.2023.102224.
  PUBMED    37071532
REFERENCE   2  (bases 1 to 8521)
  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: "STAR 
            Protoc."; date: "2023-04-17"; volume: "4(2):102224. doi"; pages: " 
            10.1016/j.xpro.2023.102224"
COMMENT     Sequence Label: pMSCV_IRES_GFP_CD16a
FEATURES             Location/Qualifiers
     source          1..8521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1476..1500
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2275..2299)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2310..2333
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2337..2360
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     2766..2785
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2854..3453
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2854..2873)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3350..3370
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3514..4079
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3529..4081
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3673..3690)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3900..3919
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4093..4095,4096..4098,4099..4812)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4138..4159)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4399..4418)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4746..4767
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4914..5428
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5590..5606
                     /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)."
     promoter        complement(join(5614..5620,5621..5638,5639..5644))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5659..5680
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5797..5814)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5968..6556)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6048..6067)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6727..7518,7519..7587))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNIGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7350..7369
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7588..7692)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7760..7778
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7816..7838)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7938..7957
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8151..8173
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8301..8320
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8520..8521,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctctcga ggttaacgaa ttctaccggg
     1441 tatagggaga cccaagctgg ctagttaagc tatcaacaag tttgtacaaa aaagcaggct
     1501 tcgccaccat gtggcagctg ctcctcccaa ctgctctgct acttctagtt tcagctggca
     1561 tgcggactga agatctccca aaggctgtgg tgttcctgga gcctcaatgg tacagggtgc
     1621 tcgagaagga cagtgtgact ctgaagtgcc agggagccta ctcccctgag gacaattcca
     1681 cacagtggtt tcacaatgag agcctcatct caagccaggc ctcgagctac ttcattgacg
     1741 ctgccacagt cgacgacagt ggagagtaca ggtgccagac aaacctctcc accctcagtg
     1801 acccggtgca gctagaagtc catatcggct ggctgttgct ccaggcccct cggtgggtgt
     1861 tcaaggagga agaccctatt cacctgaggt gtcacagctg gaagaacact gctctgcata
     1921 aggtcacata tttacagaat ggcaaaggca ggaagtattt tcatcataat tctgacttct
     1981 acattccaaa agccacactc aaagacagcg gctcctactt ctgcaggggg cttgtgggga
     2041 gtaaaaatgt gtcttcagag actgtgaaca tcaccatcac tcaaggtttg gcagtgtcaa
     2101 ccatctcatc attctttcca cctgggtacc aagtctcttt ctgcttggtg atggtactcc
     2161 tttttgcagt ggacacagga ctatatttct ctgtgaagac aaacattcga agctcaacaa
     2221 gagactggaa ggaccataaa tttaaatgga gaaaggaccc tcaagacaaa tgagacccag
     2281 ctttcttgta caaagtggtt gatctagagg attacaagga tgacgacgat aagggagatt
     2341 acaaggatga cgacgataag tgatctagag ggcccgcggt tcgaaggtaa gcctatcttg
     2401 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2461 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2521 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2581 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2641 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2701 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     2761 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     2821 tccgggcctt tcgacctgca gcccaagctt accatgaccg agtacaagcc cacggtgcgc
     2881 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2941 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3001 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3061 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     3121 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     3181 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     3241 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     3301 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     3361 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     3421 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     3481 accgaaagga gcgcacgacc ccatgcatcc aattccgccc cccccctaac gttactggcc
     3541 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3601 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3661 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     3721 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     3781 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     3841 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     3901 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     3961 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     4021 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     4081 atggccacaa ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     4141 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtctggcga gggcgagggc
     4201 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     4261 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     4321 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     4381 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     4441 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     4501 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     4561 aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga ggacggcagc
     4621 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     4681 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     4741 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     4801 ctgtacaagt aatgaattaa ttaagaatta tcaagcttat cgataccgtc gacctgcagc
     4861 caagcttatc gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     4921 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     4981 tacataactg agaatagaga agttcagatc aaggttagga acagagagac agcagaatat
     5041 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat
     5101 ggtccccaga tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg
     5161 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     5221 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     5281 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct
     5341 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     5401 tgactacccg tcagcggggg tctttcatgg gtaacagttt cttgaagttg gagaacaaca
     5461 ttctgagggt aggagtcgaa tattaagtaa tcctgactca attagccact gttttgaatc
     5521 cacatactcc aatactcctg aaatagttca ttatggacag cgcagaaaga gctggggaga
     5581 attgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     5641 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     5701 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5761 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     5821 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     5881 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     5941 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6001 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6061 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     6121 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     6181 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     6241 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     6301 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     6361 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     6421 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     6481 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     6541 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     6601 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     6661 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     6721 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     6781 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     6841 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     6901 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6961 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7021 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7081 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccaa tgttgtgcaa
     7141 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     7201 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     7261 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     7321 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     7381 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     7441 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     7501 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     7561 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     7621 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     7681 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     7741 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     7801 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     7861 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     7921 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     7981 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     8041 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     8101 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     8161 acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     8221 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     8281 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     8341 gcgccggtga tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa
     8401 gacaggatat cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct
     8461 atagagtacg agccatagat aaaataaaag attttattta gtctccagaa aaagggggga
     8521 a
//
