LOCUS       sequence-333571-       10522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for a sgRNA against BFP and spCas9-Ec86 RT fusion 
            protein linked to mCherry via a T2A peptide. Donor template An was 
            inserted in the Retron Ec86 msr-msd region by SpeI and AvrII..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10522)
  AUTHORS   Zhao B, Chen SA, Lee J, Fraser HB
  TITLE     Bacterial Retrons Enable Precise Gene Editing in Human Cells.
  JOURNAL   CRISPR J. 2022 Jan 24. doi: 10.1089/crispr.2021.0065.
  PUBMED    35076284
REFERENCE   2  (bases 1 to 10522)
  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: "CRISPR J. 
            2022 Jan 24. doi: 10.1089/crispr.2021.0065."
COMMENT     Sequence Label: 
            pBZ210-pU6-sgBFP-An_CBh-sv40NLS-Cas9-Ec86RT-NLS-T2A-mCherry
FEATURES             Location/Qualifiers
     source          1..10522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        6..254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6..26
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     177..196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        275..350
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        635..920
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        922..1199
                     /label=chicken beta-actin promoter
     intron          1200..1427
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1439..1448
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1448..1513
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1520..1540
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1565..5665
                     /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             1565..5665
                     /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             6722..6769
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6773..6826
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6833..7543
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(6978..6996)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7246..7265
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(7568..7585)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7574..7781
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7790..7930
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7790..7919
                     /label=AAV2 ITR
     rep_origin      8005..8460
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8092..8111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8302..8323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8477..8496)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8596..8618
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8656..8674)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8742..8846
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8847..8915,8916..9707)
                     /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(9065..9084)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9878..10466
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10367..10386
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
       61 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
      121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
      181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
      241 aaggacgaaa caccgctgaa gcactgcacg ccatgtttta gagctagaaa tagcaagtta
      301 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc cgtacgatgc
      361 gcacccttag cgagaggttt atcattaagg tcaacctctg gatgttgttt cggcatcctg
      421 cattgaatct gagttactgt ctgtttccct actagtaagt cgtgctgctt catgtggtcg
      481 gggtagcggc tgaagcactg cacgccgtac gtcagggtgg tcacgagggt gggccagggc
      541 acgggcagct tgccggtggt gcagatgaac ttcaggccta ggagggaacc cgtttcttct
      601 gacgtaaggg tgcgcatttt tttctagagg tacccgttac ataacttacg gtaaatggcc
      661 cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga
      721 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      781 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      841 ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      901 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      961 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
     1021 tgggggcggg gggggggggg gggcgcgcgc cggggggggg gggggggggg gggggggggg
     1081 gggggggggg gcggggaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
     1141 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
     1201 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
     1261 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
     1321 cgggctgtaa ttagctgagc aagaggtaag ggtttaaggg atggttggtt ggtggggtat
     1381 taatgtttaa ttacctggag cacctgcctg aaatcacttt ttttcaggtt ggaccggtgc
     1441 caccatggac tataaggacc acgacggaga ctacaaggat catgatattg attacaaaga
     1501 cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc
     1561 agccgacaag aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt
     1621 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
     1681 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
     1741 ggccacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
     1801 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
     1861 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
     1921 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     1981 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     2041 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     2101 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     2161 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     2221 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa
     2281 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     2341 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     2401 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     2461 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     2521 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     2581 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     2641 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     2701 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     2761 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     2821 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     2881 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     2941 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     3001 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     3061 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     3121 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     3181 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     3241 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     3301 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     3361 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     3421 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     3481 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     3541 caggctgagc cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct
     3601 ggatttcctg aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga
     3661 cagcctgacc tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct
     3721 gcacgagcac attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac
     3781 agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt
     3841 gatcgaaatg gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag
     3901 aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc
     3961 cgtggaaaac acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg
     4021 ggatatgtac gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca
     4081 tatcgtgcct cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag
     4141 cgacaagaac cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa
     4201 gaactactgg cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct
     4261 gaccaaggcc gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca
     4321 gctggtggaa acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa
     4381 cactaagtac gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc
     4441 caagctggtg tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa
     4501 ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa
     4561 gtaccctaag ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa
     4621 gatgatcgcc aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag
     4681 caacatcatg aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg
     4741 gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt
     4801 tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt
     4861 gcagacaggc ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat
     4921 cgccagaaag aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc
     4981 ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt
     5041 gaaagagctg ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga
     5101 ctttctggaa gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa
     5161 gtactccctg ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact
     5221 gcagaaggga aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag
     5281 ccactatgag aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga
     5341 acagcacaag cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt
     5401 gatcctggcc gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa
     5461 gcccatcaga gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc
     5521 ccctgccgcc ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa
     5581 agaggtgctg gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat
     5641 cgacctgtct cagctgggag gcgactctgg aggatctagc ggaggatcct ctggcagcga
     5701 gacaccagga acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag
     5761 cagcaagagt gcggaatacc ttaacacatt taggcttaga aacctgggac tccccgttat
     5821 gaataacctg cacgatatgt ctaaggcaac ccgaatcagt gtagagacgc ttagactctt
     5881 gatatatacc gccgactttc gatacagaat atataccgtc gagaagaagg gcccagagaa
     5941 acgaatgcgg accatatacc aacctagtag ggagctgaaa gcgcttcagg gctgggtact
     6001 tcgaaatatc cttgacaaac tttccagttc accgttcagc attggtttcg agaaacacca
     6061 gagtattctg aataacgcga cacctcacat aggagccaat ttcatcctca atattgacct
     6121 ggaggatttc ttccctagcc ttactgccaa taaagtgttc ggtgtattcc acagcctcgg
     6181 ctacaaccga ctgatttcat ctgtactcac aaaaatttgt tgctataaga acctgctccc
     6241 tcaaggggcc ccaagtagcc caaaactggc aaacctcatc tgttcaaaat tggactatcg
     6301 aattcagggc tatgccggat ccaggggctt gatctacacg agatacgcag acgacctgac
     6361 actttcagca caatccatga agaaagttgt taaagcgagg gattttcttt tttccattat
     6421 tccgtctgaa ggattggtta ttaattctaa aaaaacttgc attagtggtc ctcggtctca
     6481 acgaaaggtt acaggcctgg taatctctca ggagaaggtt gggattggaa gagagaagta
     6541 taaagagatt cgcgccaaaa tacatcatat tttttgcggt aaatcatctg aaatcgagca
     6601 cgtaagggga tggctttctt tcattctttc tgtagactcc aaatctcacc gacgactcat
     6661 cacatatata agtaaactgg agaaaaaata tgggaaaaat ccgcttaaca aggctaaaac
     6721 taaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagc ttgagggcag
     6781 aggaagtctg ctaacatgcg gtgacgtgga ggagaatccc ggccctgcta gcatggtgag
     6841 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     6901 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     6961 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     7021 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     7081 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     7141 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     7201 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     7261 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     7321 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     7381 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     7441 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     7501 gggccgccac tccaccggcg gcatggacga gctgtacaag tgagaattcc tagagctcgc
     7561 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     7621 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     7681 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     7741 aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta
     7801 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     7861 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     7921 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     7981 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     8041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     8101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     8161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     8221 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     8281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     8341 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     8401 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     8461 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     8521 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     8581 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     8641 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     8701 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     8761 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     8821 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     8881 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     8941 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     9001 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     9061 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     9121 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     9181 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     9241 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     9301 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     9361 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     9421 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     9481 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     9541 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     9601 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     9661 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     9721 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     9781 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     9841 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     9901 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     9961 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
    10021 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
    10081 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
    10141 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
    10201 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
    10261 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
    10321 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
    10381 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
    10441 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
    10501 ccttttgctg gccttttgct ca
//
