LOCUS       sequence-210279-        8508 bp    DNA     circular SYN 15-APR-2025
DEFINITION  H588A mutant of Cas9 from Nm8013 cloned into pET28b with a 
            C-terminal Flag tag for protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8508)
  AUTHORS   Rousseau BA, Hou Z, Gramelspacher MJ, Zhang Y
  TITLE     Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 
            System from Neisseria meningitidis.
  JOURNAL   Mol Cell. 2018 Mar 1;69(5):906-914.e4. doi: 
            10.1016/j.molcel.2018.01.025. Epub 2018 Feb 15.
  PUBMED    29456189
REFERENCE   2  (bases 1 to 8508)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2018.01.025"; journalName: "Mol Cell"; date: 
            "2018-03-1- 1"; volume: "69"; issue: "5"; pages: "906-914.e4"
COMMENT     Sequence Label: pET28b-NmCas9-Flag H588A
FEATURES             Location/Qualifiers
     source          1..8508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..37
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     121..140
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        121..139
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    140..164
                     /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)."
     RBS             179..201
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             208..3453
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             3454..3477
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3490..3510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=7xHis
                     /translation="HHHHHHH"
     primer_bind     complement(3563..3581)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3577..3624
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3661..4116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3748..3767)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3958..3979
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4208..5023)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4208..5023)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4931..4950
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5015..5034
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5145..5733
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5634..5653
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5887..5904
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5919..6058
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5984..6003)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6144..6166
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6160..6351)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7160..8242)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8204..8223
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8243..8320)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg
       61 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
      121 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
      181 tgtttaactt taagaaggag atataccatg gctgccttca aacctaattc aatcaactac
      241 atcctcggcc tcgatatcgg catcgcatcc gtcggctggg cgatggtaga aattgacgaa
      301 gaagaaaacc ccatccgcct gattgatttg ggcgtgcgcg tatttgagcg tgccgaagta
      361 ccgaaaacag gcgactccct tgccatggca aggcgtttgg cgcgcagtgt tcgccgcctg
      421 acccgccgtc gcgcccaccg cctgcttcgg acccgccgcc tattgaaacg cgaaggcgta
      481 ttacaagccg ccaattttga cgaaaacggc ttgattaaat ccttaccgaa tacaccatgg
      541 caacttcgcg cagccgcatt agaccgcaaa ctgacgcctt tagagtggtc ggcagtcttg
      601 ttgcatttaa tcaaacatcg cggctattta tcgcaacgga aaaacgaggg cgaaactgcc
      661 gataaggagc ttggcgcttt gcttaaaggc gtagccggca atgcccatgc cttacagaca
      721 ggcgatttcc gcacaccggc cgaattggct ttaaataaat ttgagaaaga aagcggccat
      781 atccgcaatc agcgcagcga ttattcgcat acgttcagcc gcaaagattt acaggcggag
      841 ctgattttgc tgtttgaaaa acaaaaagaa tttggcaatc cgcatgtttc aggcggcctt
      901 aaagaaggta ttgaaaccct actgatgacg caacgccctg ccctgtccgg cgatgccgtt
      961 caaaaaatgt tggggcattg caccttcgaa ccggcagagc cgaaagccgc taaaaacacc
     1021 tacacagccg aacgtttcat ctggctgacc aagctgaaca acctgcgtat tttagagcaa
     1081 ggcagcgagc ggccattgac cgataccgaa cgcgccacgc ttatggacga gccatacaga
     1141 aaatccaaac tgacttacgc acaagcccgt aagctgctgg gtttagaaga taccgccttt
     1201 ttcaaaggct tgcgctatgg taaagacaat gccgaagcct caacattgat ggaaatgaag
     1261 gcctaccatg ccatcagccg tgcactggaa aaagaaggat tgaaagacaa aaaatcccca
     1321 ttaaaccttt ctcccgaatt acaagacgaa atcggcacgg cattctccct gttcaaaacc
     1381 gatgaagaca ttacaggccg tctgaaagac cgtatacagc ccgaaatctt agaagcgctg
     1441 ttgaaacaca tcagcttcga taagttcgtc caaatttcct tgaaagcatt gcgccgaatt
     1501 gtgcctctaa tggaacaagg caaacgttac gatgaagcct gcgccgaaat ctacggagac
     1561 cattacggca agaagaatac ggaagaaaag atttatctgc cgccgattcc cgccgacgaa
     1621 atccgcaacc ccgtcgtctt gcgcgcctta tctcaagcac gtaaggtcat taacggcgtg
     1681 gtacgccgtt acggctcccc agctcgtatc catattgaaa ctgcaaggga agtaggtaaa
     1741 tcgtttaaag accgcaaaga aattgagaaa cgccaagaag aaaaccgcaa agaccgggaa
     1801 aaagccgccg ccaaattccg agagtatttc cccaattttg tcggagaacc caaatccaaa
     1861 gatattctga aactgcgcct gtacgagcaa caacacggca aatgcctgta ttcgggcaaa
     1921 gaaatcaact taggccgtct gaacgaaaaa ggctatgtcg aaatcgacgc tgccctgccg
     1981 ttctcgcgca catgggacga cagtttcaac aataaagtac tggtattggg cagcgaaaac
     2041 caaaacaaag gcaatcaaac cccttacgaa tacttcaacg gcaaagacaa cagccgcgaa
     2101 tggcaggaat ttaaagcgcg tgtcgaaacc agccgtttcc cgcgcagtaa aaaacaacgg
     2161 attctgctgc aaaaattcga tgaagacggc tttaaagaac gcaatctgaa cgacacgcgc
     2221 tacgtcaacc gtttcctgtg tcaatttgtt gccgaccgta tgcggctgac aggtaaaggc
     2281 aagaaacgtg tctttgcatc caacggacaa attaccaatc tgttgcgcgg cttttgggga
     2341 ttgcgcaaag tgcgtgcgga aaacgaccgc catcacgcct tggacgccgt cgtcgttgcc
     2401 tgctcgaccg ttgccatgca gcagaaaatt acccgttttg tacgctataa agagatgaac
     2461 gcgtttgacg gtaaaaccat agacaaagaa acaggagaag tgctgcatca aaaaacacac
     2521 ttcccacaac cttgggaatt tttcgcacaa gaagtcatga ttcgcgtctt cggcaaaccg
     2581 gacggcaaac ccgaattcga agaagccgat accctagaaa aactgcgcac gttgcttgcc
     2641 gaaaaattat catctcgccc cgaagccgta cacgaatacg ttacgccact gtttgtttca
     2701 cgcgcgccca atcggaagat gagcgggcaa gggcatatgg agaccgtcaa atccgccaaa
     2761 cgactggacg aaggcgtcag cgtgttgcgc gtaccgctga cacagttaaa actgaaagac
     2821 ttggaaaaaa tggtcaatcg ggagcgcgaa cctaagctat acgaagcact gaaagcacgg
     2881 ctggaagcac ataaagacga tcctgccaaa gcctttgccg agccgtttta caaatacgat
     2941 aaagcaggca accgcaccca acaggtaaaa gccgtacgcg tagagcaagt acagaaaacc
     3001 ggcgtatggg tgcgcaacca taacggtatt gccgacaacg caaccatggt gcgcgtagat
     3061 gtgtttgaga aaggcgacaa gtattatctg gtaccgattt acagttggca ggtagcgaaa
     3121 gggattttgc cggatagggc tgttgtacaa ggaaaagatg aagaagattg gcaacttatt
     3181 gatgatagtt tcaactttaa attctcatta caccctaatg atttagtcga ggttataaca
     3241 aaaaaagcta gaatgtttgg ttactttgcc agctgccatc gaggcacagg taatatcaat
     3301 atacgcattc atgatcttga tcataaaatt ggcaaaaatg gaatactgga aggtatcggc
     3361 gtcaaaaccg ccctttcatt ccaaaaatac caaattgacg aactgggcaa agaaatcaga
     3421 ccatgccgtc tgaaaaaacg cccgcctgtc cgtgattata aagatgatga tgataaatga
     3481 gcactcgagc accaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     3541 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     3601 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     3661 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3721 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3781 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3841 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3901 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3961 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     4021 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     4081 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     4141 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     4201 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     4261 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     4321 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     4381 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     4441 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     4501 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     4561 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     4621 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     4681 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     4741 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     4801 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     4861 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     4921 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     4981 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     5041 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     5101 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5161 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     5221 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     5281 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     5341 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5401 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5461 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5521 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5581 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5641 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5701 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5761 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5821 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5881 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     5941 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     6001 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     6061 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6121 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6181 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     6241 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     6301 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     6361 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     6421 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     6481 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     6541 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     6601 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     6661 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     6721 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     6781 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     6841 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     6901 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     6961 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     7021 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     7081 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     7141 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     7201 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     7261 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     7321 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     7381 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     7441 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     7501 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     7561 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     7621 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     7681 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     7741 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     7801 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     7861 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     7921 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     7981 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     8041 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     8101 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     8161 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     8221 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     8281 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     8341 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     8401 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     8461 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcga
//
