LOCUS       Exported                9618 bp ds-DNA     circular SYN 18-FEB-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9618)
  AUTHORS   Institute of Molecular and Genomic Medicine
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Feb 18, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9618
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     2..22
                     /label=T3
                     /note="T3 promoter, forward primer"
     primer_bind     5..24
                     /label=mission_T3 promoter seq
     primer_bind     complement(22..42)
                     /label=Primer 2
     primer_bind     complement(23..48)
                     /label=pp438-EcoRI-longum core-F
     primer_bind     complement(25..48)
                     /label=Primer 3
     primer_bind     42..68
                     /label=pp453-EcoRI-kasOp-F
     misc_feature    49..145
                     /label=KasOp*
     primer_bind     49..68
                     /label=pp441-SpeI-kasOP17tss-sgRNA-F
     primer_bind     complement(49..68)
                     /label=npPP111
     primer_bind     complement(90..148)
                     /label=pp144
     primer_bind     117..165
                     /label=pp457-kasOp-Cas9-F
     primer_bind     complement(117..136)
                     /label=pp454-kasOp-R
     primer_bind     complement(126..155)
                     /label=pp481-BbsI kasop-R
     primer_bind     complement(126..148)
                     /label=npPP249
     CDS             146..832
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     primer_bind     146..164
                     /label=APOBEC-F
     primer_bind     147..166
                     /label=pp483-BbsI-Cas9-F
     primer_bind     complement(287..305)
                     /label=pp494-APO_EcoRI site
     CDS             881..4981
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     1072..1082
                     /label=Primer 1
     primer_bind     1102..1123
                     /label=Primer 4
     primer_bind     complement(1126..1149)
                     /label=Cas9-R
     CDS             4994..5245
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     terminator      5426..5474
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     primer_bind     complement(5553..5581)
                     /label=pp459-Cas9-R
     primer_bind     5575..5663
                     /label=pp451-SpeI-kasOP17tss-F
     primer_bind     5575..5600
                     /label=pp441-SpeI-kasOP17tss-sgRNA-F
     misc_feature    5581..6231
                     /label=golden gate compatible sgRNA insertion cassette
     misc_feature    5581..5642
                     /label=KasOp17tss
     primer_bind     complement(5581..5600)
                     /label=npPP111
     primer_bind     5581..5600
                     /label=pp453-EcoRI-kasOp-F
     primer_bind     5643..5663
                     /label=Primer 1
     misc_feature    5645..5650
                     /label=BbsI
     CDS             complement(5651..5947)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSG"
     primer_bind     5797..5819
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5811..5828
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5838..5857
                     /label=T7
                     /note="T7 promoter, forward primer"
     primer_bind     5889..5904
                     /label=Primer 2
     primer_bind     5889..5903
                     /label=pp438-EcoRI-longum core-F
     primer_bind     5889..5901
                     /label=Primer 3
     primer_bind     complement(5902..5921)
                     /label=mission_T3 promoter seq
     primer_bind     complement(5904..5924)
                     /label=T3
                     /note="T3 promoter, forward primer"
     primer_bind     complement(5943..5964)
                     /label=mission_M13R
     primer_bind     complement(5943..5959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5956..5978)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5967..5983
                     /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(5991..6021)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6036..6057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     misc_feature    6092..6097
                     /label=BbsI
     misc_RNA        6100..6175
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      6197..6231
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     rep_origin      complement(6273..6861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6353..6372)
                     /label=pp235-pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6839..6861)
                     /label=Primer 2
     primer_bind     complement(6840..6870)
                     /label=pp440-NotI-ori-sgRNA-R
     primer_bind     6860..6890
                     /label=pp439-NotI-longum core-R
     misc_feature    6870..9618
                     /label=pMDY23 backbone
     promoter        6870..7072
                     /label=Pcat
     primer_bind     6870..6896
                     /label=pp123_IM206 primer
     CDS             7070..7729
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=chloramphenicol resistance
                     /translation="MNFNKIDLDNWKRKEIFNHYLNQQTTFSITTEIDISVLYRNIKQE
                     GYKFYPAFIFLVTRVINSNTAFRTGYNSDGELGYWDKLEPLYTIFDGVSKTFSGIWTPV
                     KNDFKEFYDLYLSDVEKYNGSGKLFPKTPIPENAFSLSIIPWTSFTGFNLNINNNSNYL
                     LPIITAGKFINKGNSIYLPLSLQVHHSVCDGYHAGLFMNSIQNCQIGLMTGFYNMR"
     primer_bind     complement(7095..7114)
                     /label=pp495_CAM-NotI site
     primer_bind     complement(7770..7824)
                     /label=pp437-mut NseI to NsiI-F
     primer_bind     complement(7829..7859)
                     /label=pp436-mut BbsI-F
     CDS             complement(8144..8461)
                     /codon_start=1
                     /transl_table=11
                     /gene="repB"
                     /product="RepB"
                     /label=repB
                     /note="pNCC293 replication protein"
                     /protein_id="ABI18631.1"
                     /translation="MVRTTLRKKRPVSARELAEAYGVSTRTIQSWVAMKREDWIDEQAA
                     MREAVRSYHDDEGHTWPQTAEHFNMSQGAVRQRCYRARKEREDEAAEKSKHLPGEIPLF
                     D"
     gene            complement(8144..8461)
                     /gene="repB"
                     /label=repB
     CDS             complement(8455..9375)
                     /codon_start=1
                     /transl_table=11
                     /gene="repA"
                     /product="RepA"
                     /label=repA
                     /note="pNCC293 replication protein"
                     /protein_id="ABI18630.1"
                     /translation="MYAVAMSDEYSQPTLELSRTFEGWWLPERPLCCDDDYSQLHRRSR
                     ADALKCKHIEANPAALVNTIVVDIDDANAKAMALWEHEGMRPNWIAENPANGHAHAGWV
                     LTFPVPRTDLARLKPLKLLHATTEGLRRSCDGDMGYSGLLMKNPEHPAWASDIIEWDTY
                     DLEQLVQSLQEHGDMPPVSWKRTKRARTQGLGRNCTLFDKARTLAYRYVRRLPDRSEAS
                     SEALRLYVRRTCHELNVSLFPDPLHAREVEDIAKSIHKWIVTRSRMWLDGAIANAATFI
                     AIQSARGHKHGENKYQQVMKEALEW"
     gene            complement(8455..9375)
                     /gene="repA"
                     /label=repA
     primer_bind     9521..9540
                     /label=pp154
     primer_bind     complement(9598..9618)
                     /label=Primer 1
ORIGIN
        1 tgcaattaac cctcactaaa gggaacaaaa gctggagctc cagaattctg ttcacattcg
       61 aacggtctct gctttgacaa catgctgtgc ggtgttgtaa agtcgtggcc aggagaatac
      121 gacagcgtgc aggactgggg gagttatgtc ctccgagacc ggccccgtgg ccgtggaccc
      181 gaccctgcgt cgccgcatcg agccgcacga gttcgaggtg ttcttcgacc cgcgcgagct
      241 gcgcaaggag acctgcctgc tgtacgagat caactggggc ggccggcact ccatctggcg
      301 ccacacctcg cagaacacca acaagcacgt cgaggtcaac ttcatcgaga agttcaccac
      361 cgagcggtac ttctgcccga acacccgctg ctccatcacc tggttcctgt cctggtcccc
      421 ctgcggcgag tgctcccggg ccatcaccga gttcctgtcc cgctacccgc acgtcaccct
      481 gttcatctac atcgcccggc tgtaccacca cgccgacccg cgcaaccggc agggcctgcg
      541 cgacctgatc tcctccggcg tcaccatcca gatcatgacc gagcaggagt ccggctactg
      601 ctggcggaac ttcgtcaact actccccgtc caacgaggcc cactggcccc gctacccgca
      661 cctgtgggtc cgcctgtacg tcctggagct gtactgcatc atcctgggcc tgccgccctg
      721 cctgaacatc ctgcgtcgca agcagccgca gctgaccttc ttcaccatcg ccctccagtc
      781 ctgccactac cagcgcctgc cgccccacat cctgtgggcc accggcctga agtccggctc
      841 cgagaccccg ggcacctcgg agtccgccac cccggagtcc gacaagaagt actccatcgg
      901 cctcgcgatc ggcaccaact ccgtgggctg ggcggtcatc accgacgagt acaaggtccc
      961 ctccaagaag ttcaaggtcc tgggcaacac cgaccggcac tcgatcaaga agaacctgat
     1021 cggcgccctg ctcttcgaca gcggcgagac cgccgaggcg acccgcctga agcggaccgc
     1081 gcgtcgccgc tacacccggc gcaagaaccg catctgctac ctgcaggaaa tcttctccaa
     1141 cgagatggcc aaggtggacg actcgttctt ccaccgcctg gaggagagct tcctggtgga
     1201 ggaggacaag aagcacgagc gccacccgat cttcggcaac atcgtggacg aggtggccta
     1261 ccacgagaag taccccacca tctaccacct ccgcaagaag ctggtggact cgaccgacaa
     1321 ggcggacctg cggctcatct acctggccct cgcgcacatg atcaagttcc gcggccactt
     1381 cctcatcgag ggcgacctga acccggacaa ctccgacgtg gacaagctct tcatccagct
     1441 ggtgcagacc tacaaccagc tgttcgagga gaaccccatc aacgccagcg gcgtggacgc
     1501 caaggcgatc ctctccgcgc gcctgagcaa gtcccggcgc ctggagaacc tcatcgccca
     1561 gctgccgggc gagaagaaga acggcctctt cggcaacctg atcgcgctgt cgctcggcct
     1621 gacccccaac ttcaagagca acttcgacct ggccgaggac gcgaagctcc agctgtccaa
     1681 ggacacctac gacgacgacc tggacaacct gctcgcccag atcggcgacc agtacgcgga
     1741 cctcttcctg gccgcgaaga acctctcgga cgccatcctg ctcagcgaca tcctgcgggt
     1801 caacaccgag atcaccaagg ccccgctgtc ggcgagcatg atcaagcggt acgacgagca
     1861 ccaccaggac ctgaccctgc tcaaggccct cgtgcgccag cagctgcccg agaagtacaa
     1921 ggaaatcttc ttcgaccagt ccaagaacgg ctacgccggc tacatcgacg gcggcgcgtc
     1981 gcaggaggag ttctacaagt tcatcaagcc gatcctggag aagatggacg gcaccgagga
     2041 gctgctcgtc aagctgaacc gcgaggacct gctccgcaag cagcggacct tcgacaacgg
     2101 ctccatcccg caccagatcc acctgggcga gctccacgcc atcctccggc gccaggagga
     2161 cttctacccc ttcctgaagg acaaccgcga gaagatcgag aagatcctga ccttccgcat
     2221 cccgtactac gtcggccccc tggcccgcgg caactcccgg ttcgcgtgga tgacccggaa
     2281 gtcggaggag accatcaccc cgtggaactt cgaggaggtc gtggacaagg gcgcgtccgc
     2341 gcagtcgttc atcgagcgca tgaccaactt cgacaagaac ctcccgaacg agaaggtcct
     2401 gcccaagcac tccctgctct acgagtactt caccgtgtac aacgagctga ccaaggtcaa
     2461 gtacgtgacc gagggcatgc ggaagccggc cttcctgtcg ggcgagcaga agaaggcgat
     2521 cgtggacctg ctcttcaaga ccaaccgcaa ggtcaccgtg aagcagctga aggaggacta
     2581 cttcaagaag atcgagtgct tcgactccgt cgagatcagc ggcgtggagg accgcttcaa
     2641 cgcctccctg ggcacctacc acgacctgct caagatcatc aaggacaagg acttcctcga
     2701 caacgaggag aacgaggaca tcctggagga catcgtcctc accctgaccc tcttcgagga
     2761 ccgcgagatg atcgaggagc ggctcaagac ctacgcccac ctgttcgacg acaaggtgat
     2821 gaagcagctg aagcgtcgcc gctacaccgg ctggggccgc ctctcccgga agctgatcaa
     2881 cggcatccgg gacaagcaga gcggcaagac catcctggac ttcctcaagt ccgacggctt
     2941 cgccaaccgc aacttcatgc agctcatcca cgacgacagc ctgaccttca aggaggacat
     3001 ccagaaggcc caggtctcgg gccagggcga cagcctccac gagcacatcg ccaacctggc
     3061 gggctccccg gcgatcaaga agggcatcct ccagaccgtc aaggtcgtgg acgagctggt
     3121 caaggtgatg ggccgccaca agcccgagaa catcgtgatc gagatggccc gggagaacca
     3181 gaccacccag aagggccaga agaactcgcg cgagcggatg aagcggatcg aggagggcat
     3241 caaggagctc ggcagccaga tcctgaagga gcacccggtc gagaacaccc agctgcagaa
     3301 cgagaagctg tacctctact acctgcagaa cggccgcgac atgtacgtgg accaggagct
     3361 cgacatcaac cggctgtccg actacgacgt ggaccacatc gtgccgcagt ccttcctgaa
     3421 ggacgactcg atcgacaaca aggtcctgac ccgctcggac aagaaccggg gcaagtccga
     3481 caacgtgccc tcggaggagg tcgtgaagaa gatgaagaac tactggcgcc agctgctcaa
     3541 cgccaagctc atcacccagc gcaagttcga caacctgacc aaggccgagc ggggcggcct
     3601 gagcgagctc gacaaggcgg gcttcatcaa gcgccagctg gtcgagaccc ggcagatcac
     3661 caagcacgtg gcccagatcc tggactcccg gatgaacacc aagtacgacg agaacgacaa
     3721 gctgatccgc gaggtcaagg tgatcaccct caagagcaag ctggtctccg acttccgcaa
     3781 ggacttccag ttctacaagg tccgggagat caacaactac caccacgccc acgacgcgta
     3841 cctgaacgcc gtcgtgggca ccgcgctgat caagaagtac ccgaagctgg agtccgagtt
     3901 cgtctacggc gactacaagg tctacgacgt gcgcaagatg atcgccaaga gcgagcagga
     3961 gatcggcaag gccaccgcga agtacttctt ctactccaac atcatgaact tcttcaagac
     4021 cgagatcacc ctggccaacg gcgagatccg caagcggccc ctgatcgaga ccaacggcga
     4081 gaccggcgag atcgtctggg acaagggccg cgacttcgcc accgtccgga aggtgctgtc
     4141 gatgccgcag gtcaacatcg tgaagaaaac cgaggtgcag accggcggct tcagcaagga
     4201 gtccatcctc cccaagcgca acagcgacaa gctgatcgcc cggaagaagg actgggaccc
     4261 gaagaagtac ggcggcttcg acagccccac cgtcgcctac tccgtgctgg tcgtggcgaa
     4321 ggtcgagaag ggcaagagca agaagctgaa gtccgtgaag gagctgctcg gcatcaccat
     4381 catggagcgc tcctcgttcg agaagaaccc gatcgacttc ctggaggcca agggctacaa
     4441 ggaggtcaag aaggacctca tcatcaagct gcccaagtac agcctgttcg agctggagaa
     4501 cggccgcaag cggatgctcg cctccgcggg cgagctgcag aagggcaacg agctggccct
     4561 cccgtcgaag tacgtcaact tcctgtacct cgcgtcccac tacgagaagc tgaagggctc
     4621 gcccgaggac aacgagcaga agcagctctt cgtggagcag cacaagcact acctggacga
     4681 gatcatcgag cagatcagcg agttcagcaa gcgcgtcatc ctggccgacg cgaacctcga
     4741 caaggtgctg tccgcctaca acaagcaccg cgacaagccg atccgggagc aggcggagaa
     4801 catcatccac ctgttcaccc tcaccaacct gggcgccccc gccgcgttca agtacttcga
     4861 caccaccatc gaccgcaagc ggtacacctc caccaaggag gtcctcgacg cgaccctgat
     4921 ccaccagagc atcaccggcc tgtacgagac ccgcatcgac ctgtcccagc tcggcggcga
     4981 ctccggcggc tccaccaacc tgtccgacat catcgagaag gagaccggca agcagctggt
     5041 catccaggag tccatcctga tgctgccgga ggaagtcgag gaagtcatcg gcaacaagcc
     5101 cgagtccgac atcctggtcc acaccgccta cgacgagtcc accgacgaga acgtcatgct
     5161 gctgacctcg gacgcccccg agtacaagcc ctgggccctg gtcatccagg actccaacgg
     5221 cgagaacaag atcaagatgc tgtgatctag aggatccccg ggtaccgagc tcggattccc
     5281 cagatctaaa gttttgtcgt ctttccagac gttagtaaat gaattttctg tatgaggttt
     5341 tgctaaacaa ctttcaacag tttcagcgga gtgagaatag aaaggaacaa ctaaaggaat
     5401 tgcgaataat aattttttca cgttgaaaat ctccaaaaaa aaaggctcca aaaggagcct
     5461 ttaattgtat cggtttatca gcttgctttc gaggtgaatt tcttaaacag cttgataccg
     5521 atagttgcgc cgacaatgac aacaaccatc gcccacgcat aaccgatata ttcgactagt
     5581 tgttcacatt cgaacggtct ctgctttgac aacatgctgt gcggtgtgta aagtcgtggc
     5641 caaagtcttc tcagccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     5701 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5761 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5821 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc
     5881 tcgaggtcct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     5941 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     6001 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     6061 gcgttgcgct cactgcccgc tttccaccgg tgaagacatg ttttagagct agaaatagca
     6121 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     6181 actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta tctagaggcc
     6241 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     6301 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     6361 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     6421 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6481 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaccccccc
     6541 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6601 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6661 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6721 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6781 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6841 cgcagaaaaa aaggatctca agcggccgca tcccattatg ctttggcagt ttattcttga
     6901 catgtagtga gggggctggt ataatcacat acggccgata aagcaagcat ataatattgc
     6961 gtttcatctt tagaagcgaa tttcgccaat attataatta tcaaagagag gggtggcaaa
     7021 cggtatttgg cattattagg ttaaaaaatg tagaaggaga gtgaaaccca tgaactttaa
     7081 taaaattgat ttagacaatt ggaagagaaa agagatattt aatcattatt tgaaccaaca
     7141 aacgactttt agtataacca cagaaattga tattagtgtt ttataccgaa acataaaaca
     7201 agaaggatat aaattttacc ctgcatttat tttcttagtg acaagggtga taaactcaaa
     7261 tacagctttt agaactggtt acaatagcga cggagagtta ggttattggg ataagttaga
     7321 gccactttat acaatttttg atggtgtatc taaaacattc tctggtattt ggactcctgt
     7381 aaagaatgac ttcaaagagt tttatgattt atacctttct gatgtagaga aatataatgg
     7441 ttcggggaaa ttgtttccca aaacacctat acctgaaaat gctttttctc tttctattat
     7501 tccatggact tcatttactg ggtttaactt aaatatcaat aataatagta attaccttct
     7561 acccattatt acagcaggaa aattcattaa taaaggtaat tcaatatatt taccgctatc
     7621 tttacaggta catcattctg tttgtgatgg ttatcatgca ggattgttta tgaactctat
     7681 tcagaattgt cagataggcc taatgactgg cttttataat atgagataat gccgactgta
     7741 ctttttacag tcggttttct aatgtcacta acctgccccg ttagttgatg catgatcatc
     7801 ataattctgt ctcattatct agagtcgcca ttggggaaga agagcaggat ttgcctgcgg
     7861 ttgaccgaga cggagtgggc gacgatgaac cggatggcgt tcgcgtgcgg tcagatggat
     7921 atctccatgg acacccacat ggatatctcc atggacaccc acatggatat ctccatcaaa
     7981 tcgaaggtaa gcaagctaga cggttcatgt acatgaaccg aacgcagcac ctccctcgcc
     8041 ttgcggcgct ggagcaacaa cccgagtgcc gaaggccgat ggaagaggaa aaagccgcaa
     8101 ggcgagggag gtgctgcgtt cgcgtttggg acaacgttta gcgtcagtcg aacagtggaa
     8161 tctcgccggg tagatgcttc gatttctccg ccgcctcgtc ctcgcgctcc ttgcgagccc
     8221 tgtagcagcg ttgacgcacg gcaccctggc tcatgttgaa atgctcggcg gtctgcggcc
     8281 atgtatggcc ctcgtcatcg tgatatgagc ggactgcttc gcgcatagcg gcttgttcat
     8341 caatccaatc ctcgcgcttc attgccaccc agctctgaat ggtgcgcgtg gagacgccgt
     8401 atgcttcagc taattcacgt gcagacaccg ggcgcttctt cctcaaagtc gtccttacca
     8461 ttccagtgcc tccttcatga cctgctgata tttgttctca ccgtgtttgt gtcctcgtgc
     8521 ggattggatg gcgatgaatg tggctgcgtt ggcaatggca ccgtcgagcc acatgcggct
     8581 gcgggtgacg atccatttgt ggatgctctt ggcgatgtcc tcgacctcac gcgcgtgcag
     8641 cggatcgggg aacagcgaga cgttgagttc gtggcaggtg cggcgcacgt ataggcgcaa
     8701 tgcctcgctg ctggcctccg aacggtcggg cagccgccgc acgtagcggt aggcgagcgt
     8761 gcgggccttg tcgaagagcg tgcagttgcg tcccagcccc tgcgtgcggg cgcgcttggt
     8821 gcgcttccag ctgacgggcg gcatgtcccc gtgttcctgg agcgactgca cgagctgttc
     8881 caggtcgtag gtgtcccact cgatgatgtc cgacgcccac gccggatgct cggggttctt
     8941 catcagaagt cccgaatagc ccatgtcccc gtcgcaggag cggcgcagtc cctccgtggt
     9001 ggcgtgcagg agcttcaacg gcttgagacg cgccagatcg gttctgggca ccggaaaggt
     9061 gagcacccag cccgcgtgag cgtgcccgtt ggccgggttc tccgcgatcc agttcggccg
     9121 catgccctcg tgctcccaca gggccatcgc cttggcgttc gcgtcgtcga tgtccaccac
     9181 gatcgtgttc accagcgcgg cggggttcgc ctcgatgtgc ttgcatttga gcgcgtcggc
     9241 gcggctcctg cggtgcagct gggagtagtc gtcgtcgcag cacagcgggc gttcgggcag
     9301 ccaccagcct tcgaacgtgc gcgacagctc aagcgtcggc tgcgaatact catcggacat
     9361 ggctacagca tacatcgtat ttgtacagtg aacaaatacg cgggaaccaa gtcagatatt
     9421 tcagcccagc gaagcgtcgg ctgaacccag cccagcacat cctgcacgaa aagccgctgg
     9481 acggccctca gccgcttctc cacgccgctc acgtatgcgc gagcctctct gacctgttcg
     9541 gccgcgtgga ggccactcac ggacgtttct gagccgtctg agccgtcggc gtccgctgcg
     9601 cgttccgcag gctgaggc
//
