LOCUS       sequence-153290-        8485 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA to knock out expression of MGAT1 gene. The product of this gene
            is essential for synthesis of complex and hybrid N-Glycans..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8485)
  AUTHORS   Stolfa G, Mondal N, Zhu Y, Yu X, Buffone A Jr, Neelamegham S
  TITLE     Using CRISPR-Cas9 to quantify the contributions of O-glycans, 
            N-glycans and Glycosphingolipids to human leukocyte-endothelium 
            adhesion.
  JOURNAL   Sci Rep. 2016 Jul 26;6:30392. doi: 10.1038/srep30392.
  PUBMED    27458028
REFERENCE   2  (bases 1 to 8485)
  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: "Sci Rep."; 
            date: "2016-07-26"; volume: "6:30392. doi"; pages: " 
            10.1038/srep30392"
COMMENT     Sequence Label: pX330-MGAT1-KO
FEATURES             Location/Qualifiers
     source          1..8485
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        269..344
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        441..726
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        728..984
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          985..1212
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1224..1233
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1233..1298
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1305..1325
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1350..5450
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1350..5450
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5451..5498
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5526..5543)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5532..5739
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5748..5888
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5748..5877
                     /label=AAV2 ITR
     rep_origin      5963..6418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6050..6069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6260..6281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6435..6454)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6554..6576
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6614..6632)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6700..6804
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6805..6873,6874..7665)
                     /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(7023..7042)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7836..8424
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8325..8344
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tggggcgcta tcctctttgt tttagagcta gaaatagcaa gttaaaataa
      301 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gttttagagc
      361 tagaaatagc aagttaaaat aaggctagtc cgtttttagc gcgtgcgcca attctgcaga
      421 caaatggctc tagaggtacc cgttacataa cttacggtaa atggcccgcc tggctgaccg
      481 cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt
      541 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      601 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag
      661 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      721 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
      781 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
      841 gggggggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat
      901 cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg gcggccctat
      961 aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc
     1021 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg
     1081 agcgggcggg acggcccttc tcctccgggc tgtaattagc tgagcaagag gtaagggttt
     1141 aagggatggt tggttggtgg ggtattaatg tttaattacc tggagcacct gcctgaaatc
     1201 actttttttc aggttggacc ggtgccacca tggactataa ggaccacgac ggagactaca
     1261 aggatcatga tattgattac aaagacgatg acgataagat ggccccaaag aagaagcgga
     1321 aggtcggtat ccacggagtc ccagcagccg acaagaagta cagcatcggc ctggacatcg
     1381 gcaccaactc tgtgggctgg gccgtgatca ccgacgagta caaggtgccc agcaagaaat
     1441 tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa gaacctgatc ggagccctgc
     1501 tgttcgacag cggcgaaaca gccgaggcca cccggctgaa gagaaccgcc agaagaagat
     1561 acaccagacg gaagaaccgg atctgctatc tgcaagagat cttcagcaac gagatggcca
     1621 aggtggacga cagcttcttc cacagactgg aagagtcctt cctggtggaa gaggataaga
     1681 agcacgagcg gcaccccatc ttcggcaaca tcgtggacga ggtggcctac cacgagaagt
     1741 accccaccat ctaccacctg agaaagaaac tggtggacag caccgacaag gccgacctgc
     1801 ggctgatcta tctggccctg gcccacatga tcaagttccg gggccacttc ctgatcgagg
     1861 gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt catccagctg gtgcagacct
     1921 acaaccagct gttcgaggaa aaccccatca acgccagcgg cgtggacgcc aaggccatcc
     1981 tgtctgccag actgagcaag agcagacggc tggaaaatct gatcgcccag ctgcccggcg
     2041 agaagaagaa tggcctgttc ggaaacctga ttgccctgag cctgggcctg acccccaact
     2101 tcaagagcaa cttcgacctg gccgaggatg ccaaactgca gctgagcaag gacacctacg
     2161 acgacgacct ggacaacctg ctggcccaga tcggcgacca gtacgccgac ctgtttctgg
     2221 ccgccaagaa cctgtccgac gccatcctgc tgagcgacat cctgagagtg aacaccgaga
     2281 tcaccaaggc ccccctgagc gcctctatga tcaagagata cgacgagcac caccaggacc
     2341 tgaccctgct gaaagctctc gtgcggcagc agctgcctga gaagtacaaa gagattttct
     2401 tcgaccagag caagaacggc tacgccggct acattgacgg cggagccagc caggaagagt
     2461 tctacaagtt catcaagccc atcctggaaa agatggacgg caccgaggaa ctgctcgtga
     2521 agctgaacag agaggacctg ctgcggaagc agcggacctt cgacaacggc agcatccccc
     2581 accagatcca cctgggagag ctgcacgcca ttctgcggcg gcaggaagat ttttacccat
     2641 tcctgaagga caaccgggaa aagatcgaga agatcctgac cttccgcatc ccctactacg
     2701 tgggccctct ggccagggga aacagcagat tcgcctggat gaccagaaag agcgaggaaa
     2761 ccatcacccc ctggaacttc gaggaagtgg tggacaaggg cgcttccgcc cagagcttca
     2821 tcgagcggat gaccaacttc gataagaacc tgcccaacga gaaggtgctg cccaagcaca
     2881 gcctgctgta cgagtacttc accgtgtata acgagctgac caaagtgaaa tacgtgaccg
     2941 agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc
     3001 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa
     3061 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg
     3121 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa
     3181 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga
     3241 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga
     3301 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg
     3361 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa
     3421 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc
     3481 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg
     3541 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg
     3601 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga
     3661 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg
     3721 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt
     3781 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc
     3841 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca
     3901 tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg caagagcgac aacgtgccct
     3961 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga
     4021 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg
     4081 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg
     4141 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg
     4201 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt
     4261 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg
     4321 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg
     4381 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg
     4441 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc
     4501 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga
     4561 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag
     4621 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc
     4681 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg
     4741 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg
     4801 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa
     4861 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa
     4921 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga
     4981 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat
     5041 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata
     5101 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc
     5161 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt
     5221 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc
     5281 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg
     5341 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca
     5401 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac aaaaggccgg
     5461 cggccacgaa aaaggccggc caggcaaaaa agaaaaagta agaattccta gagctcgctg
     5521 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5581 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5641 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5701 gggggaggat tgggaagaga atagcaggca tgctggggag cggccgcagg aacccctagt
     5761 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     5821 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     5881 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5941 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6001 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     6061 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6121 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     6181 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     6241 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     6301 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     6361 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     6421 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     6481 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6541 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6601 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     6661 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6721 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6781 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     6841 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6901 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6961 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     7021 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     7081 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     7141 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     7201 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     7261 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     7321 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     7381 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     7441 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     7501 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     7561 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     7621 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     7681 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     7741 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     7801 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     7861 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7921 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7981 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     8041 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     8101 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     8161 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     8221 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     8281 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     8341 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     8401 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     8461 ttttgctggc cttttgctca catgt
//
