LOCUS       sequence-454995-       10418 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Using ef-1a promoter, expresses dCas9 and RecT protein that intended
            to be tethered via MS2 gRNA hairpin to dCas9, and Blasticidin 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10418)
  AUTHORS   Oliynyk RT, Mahas A, Karpinski E, Church GM
  TITLE     Plasmid2MC: efficient cell-free generation of high-purity minicircle
            DNA for genome editing in mammalian cells
  JOURNAL   Commun Biol 8 (2025). https://doi.org/10.1038/s42003-025-09157-7
REFERENCE   2  (bases 1 to 10418)
  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: "Commun Biol
            8 (2025). https://doi.org/10.1038/s42003-025-09157-7"
COMMENT     Sequence Label: p-att-ef1a-MS2-RecT-dCas9-BSD
FEATURES             Location/Qualifiers
     source          1..10418
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    664..697
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        806..1984
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1037..1975
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1932..1952
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1991..2000
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2000..2386
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             3317..3337
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3353..3406
                     /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             3416..3481
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3488..3508
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3533..3556,3557..3559,3560..6115,6116..6118,
                     6119..7633)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(3533..3556,3557..3559,3560..6046,6047..6049,
                     6050..7633)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7634..7681
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7691..7747
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             7748..8146
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(8174..8191)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8180..8387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8546..9001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8633..8652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8843..8864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9018..9037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     9137..9159
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(9197..9215)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9283..9387
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(9388..9456,9457..10248)
                     /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(9606..9625)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacgaga ctcagcggcc
      661 gcggtgccag ggcgtgccct tgggctcccc gggcgcgact agtgaattca gatctgatat
      721 ctctgtcggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      781 ctcaattagt cagcaaccat agtccggctc cggtgcccgt cagtgggcag agcgcacatc
      841 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
      901 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
      961 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     1021 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     1081 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     1141 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     1201 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     1261 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     1321 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     1381 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     1441 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     1501 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     1561 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     1621 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     1681 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     1741 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     1801 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     1861 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     1921 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     1981 gtgacgtacg gccaccatgg cttcaaactt tactcagttc gtgctcgtgg acaatggtgg
     2041 gacaggggat gtgacagtgg ctccttctaa tttcgctaat ggggtggcag agtggatcag
     2101 ctccaactca cggagccagg cctacaaggt gacatgcagc gtcaggcagt ctagtgccca
     2161 gaagagaaag tataccatca aggtggaggt ccccaaagtg gctacccaga cagtgggcgg
     2221 agtcgaactg cctgtcgccg cttggaggtc ctacctgaac atggagctca ctatcccaat
     2281 tttcgctacc aattctgact gtgaactcat cgtgaaggca atgcaggggc tcctcaaaga
     2341 cggtaatcct atcccttccg ccatcgccgc taactcaggt atctacagcg cttccggagg
     2401 atctagcgga ggctcctctg gctctgagac acctggcaca agcgagagcg caacacctga
     2461 aagcagcggg ggcagcagcg gggggtcagg aggatccatg acaaaacaac ctccaatagc
     2521 aaaagctgat ctgcagaaaa cccagggcaa tagagcccct gctgctgtga agaatagtga
     2581 cgtgattagc ttcatcaacc agcctagcat gaaggagcag ctggccgccg ccctgcctcg
     2641 ccacatgacc gcagagcgga tgattcggat cgctacaacc gagatcagaa aagtgcccgc
     2701 tctggggaac tgtgatacca tgtctttcgt gagcgccatt gtgcagtgta gccagctggg
     2761 cctggagccc ggaagtgccc tgggccacgc ctacctgctg cccttcggca acaagaacga
     2821 gaaatccggt aaaaagaacg tgcagctgat catcggttat agaggtatga tcgatctggc
     2881 tcggcggtcc ggccaaatcg ccagcctgtc tgccagggtg gtgagagagg gcgacgagtt
     2941 cagctttgag tttggcctgg acgaaaagtt gatccatcgg cccggagaga acgaggacgc
     3001 ccctgtcacc cacgtgtatg ccgtggctag actgaaggac ggcggtacac agttcgaagt
     3061 tatgacccgg aagcagatcg aactggtgag gtccctgtcc aaggccggca ataacgggcc
     3121 ctgggtgacc cactgggaag aaatggccaa gaaaaccgcc attagacgcc tgttcaagta
     3181 cctgcccgtg tccattgaga ttcagagggc cgtgtctatg gatgagaaag agcccctgac
     3241 catcgaccct gctgacagca gcgtgctgac cggcgagtac tctgtgattg acaattctga
     3301 ggagtctggt ggttctccta agaaaaagag gaaggtggaa ttcggcagtg gagagggcag
     3361 aggaagtctg ctaacatgcg gtgacgtgga ggagaatccc ggccctgcta gcatggacta
     3421 taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa
     3481 gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa
     3541 gtacagcatc ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga
     3601 gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa
     3661 gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct
     3721 gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga
     3781 gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc
     3841 cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga
     3901 cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga
     3961 cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt
     4021 ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct
     4081 gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag
     4141 cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa
     4201 tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggcaacc tgattgccct
     4261 gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact
     4321 gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga
     4381 ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga
     4441 catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag
     4501 atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc
     4561 tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga
     4621 cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga
     4681 cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac
     4741 cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg
     4801 gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct
     4861 gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg
     4921 gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa
     4981 gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa
     5041 cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct
     5101 gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca
     5161 gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct
     5221 gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga
     5281 agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa
     5341 ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac
     5401 actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga
     5461 cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg
     5521 gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa
     5581 gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt
     5641 taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat
     5701 tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt
     5761 ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc
     5821 cagagagaat cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat
     5881 cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac
     5941 ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt
     6001 ggaccaggaa ctggacatca accggctgtc cgactacgat gtggacgcca tcgtgcctca
     6061 gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg
     6121 gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg
     6181 gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga
     6241 gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac
     6301 ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga
     6361 cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc
     6421 cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc
     6481 ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct
     6541 ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa
     6601 gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa
     6661 ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga
     6721 gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg
     6781 gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg
     6841 cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa
     6901 ggactgggac cctaagaagt atggcggctt cgacagcccc accgtggcct attctgtgct
     6961 ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct
     7021 ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc
     7081 caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt
     7141 cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa
     7201 cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa
     7261 gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca
     7321 ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga
     7381 cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga
     7441 gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt
     7501 caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga
     7561 cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca
     7621 gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     7681 gggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     7741 tggacctatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa gagcaacggc
     7801 tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag ctctctctag
     7861 cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac cttgtgcaga
     7921 actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt gtatcgtcgc
     7981 gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac aggtgcttct
     8041 cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc cgacggcagt
     8101 tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaagaat tctaactaga
     8161 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     8221 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     8281 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     8341 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggacct gcaggagctc
     8401 gtcgacccat gggggcccgc cccaactggg gtaacctttg agttctctca gttgggggta
     8461 atcagcatca tgatgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     8521 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     8581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     8641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     8701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     8761 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     8821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     8881 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     8941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     9001 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaaacca gccccgacac
     9061 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     9121 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     9181 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     9241 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     9301 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     9361 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     9421 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     9481 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     9541 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     9601 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     9661 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     9721 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     9781 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     9841 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     9901 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     9961 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
    10021 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
    10081 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
    10141 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
    10201 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
    10261 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
    10321 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
    10381 tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
