LOCUS       sequence-430375-       14602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows for stable expression of recombinant (tagged/untagged) 
            proteins in AAVS1 locus of Human cells and transient Protein 
            Expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14602)
  TITLE     Mammalian and bacterial dual expression vector for Recombinant 
            Protein production from mammalian and Bacterial Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 14602)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pMamCol1
FEATURES             Location/Qualifiers
     source          1..14602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     terminator      99..185
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      277..304
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     polyA_signal    386..434
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(1349..1369)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2429..2443)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             complement(3770..5488)
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     regulatory      5485..5494
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          5506..5733
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(5734..6010)
                     /label=chicken beta-actin promoter
     misc_RNA        complement(6406..6481)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    6630..7466
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     terminator      complement(7532..7559)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     polyA_signal    7562..7610
                     /note="synthetic polyadenylation signal"
     CDS             complement(7627..7650)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             complement(7660..7674)
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     protein_bind    8429..8445
                     /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)."
     regulatory      8468..8477
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        8499..8950
                     /label=TRE3G BI promoter
                     /note="3rd-generation Tet-responsive bidirectional promoter
                     that can be activated by binding of Tet-On(R) 3G"
     protein_bind    8635..8653
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8671..8689
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8707..8725
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8743..8761
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8779..8797
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8815..8833
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8851..8869
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     regulatory      8974..8983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             9688..9741
                     /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             9742..10488
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    10495..10576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10532..10551)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      complement(10583..10614)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(10731..11756)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(11769..11822)
                     /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             complement(11814..11825)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    11858..11883
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    11890..12693
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     terminator      complement(12740..12834)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      complement(12914..13502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12994..13013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(13680..14489)
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     14403..14422
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     14481..14500
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
      121 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      181 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
      241 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
      301 tttttgcgtt tctacaaact ctctctggct aactagagaa cccactgctt actggcttat
      361 cgaaatgcta gcttaatcat gatttgaaca aacgacccaa cacccgtgcg ttttattctg
      421 tctttttatt gccgtaatca agcgctcatc attcgaaagg ttgtttcttg tggttccctt
      481 ccagacagtg gtacagttcg tctcccaggg ggctcatagg cccctgggtt tcagattcct
      541 cctgtactcc caggcccagg gtctcggcca caactgtagc caggttgaag ctgctcttgt
      601 caggtgtgta gctactggtg ccgtgagcct tggccatagg agactggctc tgatcgtagg
      661 tgtcggccac caggatctcg ttctcgttgg ggttgtgctg ggggtgggtg cttgatttgg
      721 acacttttct catctcattg gcgcacacag agtgttccag ctttgtgtga gtttgttcga
      781 tatatctcac gtgggggttc tcctttcttc tcagccgatt cacgccgctg aaagaaaagg
      841 ctgtgatagg agagtcgggg atcacatctt cctcgcactc cagctcggcg gcctggtgct
      901 gctggtcgtt ttcgatcttc tgctgcagct gttcggagag tttcttgttt tcttcttgca
      961 gggtgttccg ctcgttcatc agctcggtaa tcagcttcag attttgttgc ctgatattct
     1021 cgaactcttg ctgtttcttt ctcatatgtt cctcggtcac ggcgcacctg tcacataatc
     1081 cggcccgcag tctatcctcc agaaccttga tagcctcgtg aaggaccttc tgctgttctc
     1141 tcagctgctg gttcttggtg aagaactctt ccaatctctg ggcgtccagg attctctcct
     1201 gcttcagttt tgtcaccttc acttggaggc cctgcacttc tctgtcgtga cactctttca
     1261 gcttggtcca caagtccttg aagtcggagg aagtgtcggc gctgtttggg ctgccacagc
     1321 tagagccgct gatattcatt ccagcgctca ccttccgctt cttcttaggt ccggcggccg
     1381 cgccgtctcc gccgagttga gacaggtcga tccgtgtctc gtagaggccg gtgatagact
     1441 ggtggataag tgtggcgtct agcacttcct ttgtagaggt gtaccgtttt ctgtcgattg
     1501 tggtgtcgaa gtacttgaag gccgcagggg cacccaggtt ggtcagggtg aacaggtgaa
     1561 taatattttc ggcctgctcc cgtataggct tatcccggtg tttgttgtag gcagagagca
     1621 ccttgtccag gttggcgtca gccaggataa cccgtttgct gaactcgctg atctgttcga
     1681 tgatctcgtc caggtagtgc ttgtgctgtt ccacaaacag ctgtttctgt tcgttgtcct
     1741 cagggctgcc cttcagcttc tcgtagtggc tggccagata caggaagttc acatacttgc
     1801 taggcagagc cagctcgttc cccttctgca gctctccggc ggaggccagc atccgcttcc
     1861 tgccgttttc cagctcgaac aggctgtact tgggcagttt gataatcagg tccttcttca
     1921 cttccttgta gcctttggct tccaggaagt cgataggatt cttctcgaag ctgctccgtt
     1981 ccataatagt gattcccagc agctccttga cgctcttcaa tttcttgctc ttgccctttt
     2041 ctaccttggc cacgacgagg acgctgtaag ccacggtcgg agagtcaaat ccgccgtatt
     2101 tcttaggatc ccagtccttc tttctagcga tcagcttatc gctgtttctc tttggcagga
     2161 tagactcctt gctgaagcct ccagtctgca cttcggtttt cttcacaatg ttcacctgag
     2221 gcatagacag cactttccgc actgtggcaa agtctctacc cttgtcccac acgatctctc
     2281 cggtctcgcc gtttgtctct atcaggggcc tcttcctgat ttctccgttg gccagtgtaa
     2341 tttctgtctt gaagaagttc ataatgttgc tgtagaagaa gtacttggct gtagcgtcgc
     2401 cgatatcctg ctcacttttg gcaatcatct tgtcgtcgtc atccaccttg tcgtcgccgt
     2461 ccacaaactc gctttccagc ttgggatact tcttaatcag ggctgttccc accacggcgt
     2521 tcagataggc gtcgtgggcg tggtggtagt tgttaatctc ccgaaccttg tagaactgga
     2581 agtcttttct aaaatcgctc accagtttag atttcagggt gataaccttc acctctcgga
     2641 tcagcttgtc gttctcatcg tacttggtgt tcatcctgct gtccaggatc tgagcaacgt
     2701 gcttggtaat ctgcctggtc tccacaagct ggcgtttgat aaacccagcc ttgtccagct
     2761 cgctcaggcc gcctctctcg gccttggtca gattgtcaaa ctttctctgg gtaatcagct
     2821 tggcgttcag cagctgccgc cagtagttct tcatcttttt gaccacttcc tcgctgggca
     2881 cgttatcgga cttgccccgg tttttgtcgc tccgtgtcag caccttgtta tcgatactgt
     2941 cgtctttcaa gaagctctgg ggcacgatgt ggtccacgtc gtagtcggac agtctgttga
     3001 tgtccagctc ttgatccacg tacatatccc tgccgttctg caggtagtac aggtacagct
     3061 tctcgttctg cagctgggta ttttccacag ggtgttcctt caggatctgg ctgcccagtt
     3121 ccttgattcc ctcctcgatt ctcttcatcc tttctctgct gttcttctgg cccttctggg
     3181 ttgtctggtt ctcccgggcc atctcgatca cgatattctc aggcttgtgc ctgcccataa
     3241 ccttcaccag ctcgtccacg accttcactg tctgaaggat ccctttcttg atagcagggc
     3301 ttccggccag gttagcgata tgctcgtgca gtgaatcgcc ctgtccagac acttgagcct
     3361 tctgaatatc ctccttgaag gtcagagagt catcgtgaat cagctgcata aagtttctat
     3421 tggcaaagcc gtcgctcttc aggaaatcca ggatagtctt tccactctgc ttatctctga
     3481 ttccgttaat cagtttccga gataagcggc cccaccctgt atatcttctt ctcttgagct
     3541 gtttcatcac cttatcatcg aacaggtgcg cgtaggtctt cagccgttct tcgatcatct
     3601 ctctgtcctc gaacagggta agcgtcagca cgatatcctc caggatatct tcgttttcct
     3661 cgttatcgag gaagtcctta tccttgataa tcttcagcag atcgtggtat gtgccgagac
     3721 tggcgttgaa tctgtcctcc acgccgctga tctccacaga gtcgaagcac tcgatcttct
     3781 taaagtaatc ctccttcagc tgcttaacgg tcacctttct gttggtcttg aacaacagat
     3841 ccacgatagc tttcttctgc tcaccggaca ggaaggcggg ctttctcatt ccttcggtca
     3901 cgtacttgac ctttgtcagc tcgttataca ctgtgaagta ctcgtacagg aggctgtgct
     3961 taggcagcac tttctcgttg gggaggttct tgtcgaagtt ggtcatccgc tcgataaagc
     4021 tctgggcaga ggcgccctta tccacgactt cctcgaaatt ccaaggggtg attgtctctt
     4081 cgctcttccg ggtcatccag gcgaatctgc tgttgcctct ggccagaggg cccacgtagt
     4141 aggggattct gaaggtcaga attttttcga ttttctctct gttatctttc aggaacgggt
     4201 aaaaatcctc ctgccgtctc aggattgcgt ggagttcgcc caggtggatc tggtggggga
     4261 tagaaccgtt atcaaaagtt ctctgctttc tcagcaggtc ctccctgttc agcttcacca
     4321 gcagttcctc ggtgccatcc atcttttcca ggataggctt gatgaatttg tagaactcct
     4381 cctgggaggc gccgccgtcg atgtatccgg cgtagccgtt cttgctctgg tcaaagaaga
     4441 tttccttata cttttctggc agttgctgtc tgaccagggc tttcagcagc gtcagatcct
     4501 ggtggtgctc gtcgtaccgc ttaatcatag aggcgctcag aggggctttg gtgatctcgg
     4561 tgttcactct caggatatcg ctcagcaaga tagcgtcgct caggttcttg gcagccagga
     4621 acaggtcggc gtactgatct ccgatctggg ccagcaggtt gtccaggtcg tcgtcgtagg
     4681 tatctttaga cagctgcagc ttagcgtctt ctgccaggtc gaaattgctc ttgaagttgg
     4741 gggtcaggcc caggctcagg gcgataaggt tgccaaacag tccgttcttc ttctcgccag
     4801 ggagctgagc gatcaaattc tcaagtcttc tgctctttga cagtctggca gagagaatag
     4861 ccttggcgtc cactccggaa gcgttgatgg ggttctcctc aaacagctga ttgtaggtct
     4921 gaaccagctg gatgaatagc ttgtccacgt cgctgttatc agggtttagg tcgccctcga
     4981 tcagaaagtg accccggaac ttaatcatgt gggcaagggc caggtagatc agtctcagat
     5041 ctgctttgtc ggtgctatcg acgagcttct ttctcaggtg gtagattgta gggtatttct
     5101 cgtggtaggc cacttcgtcc acgatattgc cgaagattgg gtggcgctcg tgcttcttgt
     5161 cttcctcgac taggaaagat tcctccagtc tgtggaagaa gctatcgtcc accttggcca
     5221 tctcgttgct gaagatttcc tgcaggtagc aaattctatt cttccgtctt gtgtatctcc
     5281 gccgagcagt tctcttcagt ctggtggctt cggccgtctc gccggagtcg aacagcaatg
     5341 cgccgatcag gtttttcttg atagagtgtc tgtctgtgtt tcccagaacc ttgaacttct
     5401 tagaggggac cttgtattcg tcggtaatca cggcccagcc cacactgttt gtgccgatat
     5461 ccaggccgat agagtacttc ttgtccatgg cggcaccggt ctaacctgaa aaaaagtgat
     5521 ttcaggctcg tgctccaggt aattaaactt aaatacccca ccaaccaacc atcccttaaa
     5581 cccttacctc ttgctcagct aattacagcc cggaggagaa gggccgtccc gcccgctcac
     5641 ctgtgggagt aacgcggtca gtcagagccg gggcgggcgg cgcgaggcgg cggcggagcg
     5701 gggcacgggg cgaaggcagc gcgcagcgac tcccgcccgc cgcgcgcttc gctttttata
     5761 gggccgccgc cgccgccgcc tcgccagaaa aggaaacttt cggagcgcgc cgctctgatt
     5821 ggctgccgcc gcacctctcc gcctcgcccc gccccgcccc tcgccccgcc ccgccccgcc
     5881 tggcgcgcgc cccccccccc ccccgccccc aacgctgcac aaaataatta aaaaataaat
     5941 aaatacaaaa ttgggggtgg ggaggggggg gaggtgggga gagtgaagca gaacgtgggg
     6001 ctcacctcga tcaatcaatg gtaatagcga tgactaatac gtagaggtac tgccaagtag
     6061 gaaagtccca caaggtcagg tactgggcac aattccaggc gggcctctta ccgtcagtga
     6121 cgtcaatagg gggcgtactt ggcgtctgat acacttgctg tactgccaag tgggcagttt
     6181 accgtaaata ctccaccctt tgacgtcaaa ggaaagtccc tattggcgtt actattgacg
     6241 tcaacgggcg ggggtcgttg ggcggtcagc caggcgggcc tcttaccgta agttatgtaa
     6301 cgaagcttgg ccaccctggc caccagttgt ctgcagaatt ggcgcacgcg ctaaaaacgg
     6361 actagcctta ttttaacttg ctatttctag ctctaaaaca aaaaagcacc gactcggtgc
     6421 cactttttca agttgataac ggactagcct tattttaact tgctatttct agctctaaaa
     6481 catcctgtcc ctagtggccc cggcctttta ggcctttcac aagatatata aagccaagaa
     6541 atcgaaatac tttcaagtta cggtaagcta tcgtatatgc aaatatgaag gaataatggg
     6601 aaataggccc tcctgagctc ccgcggtcaa gagcagagcc aggaacccct gtagggaagg
     6661 ggcaggagag ccaggggcat gagatggtgg acgaggaagg gggacaggga agcctgagcg
     6721 cctctcctgg gcttgccaag gactcaaacc cagaagccca gagcagggcc ttagggaagc
     6781 gggaccctgc tctgggcgga ggaatatgtc ccagatagca ctggggactc tttaaggaaa
     6841 gaaggatgga gaaagagaaa gggagtagag gcggccacga cctggtgaac acctaggacg
     6901 caccattctc acaaagggag ttttccacac ggacaccccc ctcctcacca cagccctgcc
     6961 aggacggggc tggctactgg ccttatctca caggtaaaac tgacgcacgg aggaacaata
     7021 taaattgggg actagaaagg tgaagagcca aagttagaac tcaggaccaa cttattctga
     7081 ttttgttttt ccaaactgct tctcctcttg ggaagtgtaa ggaagctgca gcaccaggat
     7141 cagtgaaacg caccagacag ccgcgtcaga gcagctcagg ttctgggaga gggtagcgca
     7201 gggtggccac tgagaaccgg gcaggtcacg catccccccc ttccctccca ccccctgcca
     7261 agctctccct cccaggatcc tctctggctc catcgtaagc aaaccttaga ggttctggca
     7321 aggagagaga tggctccagg aaatgggggt gtgtcaccag ataaggaatc tgcctaacag
     7381 gaggtggggg ttagacccaa tatcaggaga ctaggaagga ggaggcctaa ggatggggct
     7441 tttctgtcac caatcctgtc cctagtgtcg acccatggtg ttggtttgat ttgtccaaac
     7501 tctgtatctt aacctcaagc cacaacactg gcgcagaaag gcccacccga aggtgagccg
     7561 acacacaaaa aaccaacaca cagatctaat gaaaataaag atcttttatt aaactcagcc
     7621 ggctcacttc tcaaactgag ggtgggacca ggccgaaccc cagtggtgag agccggctcc
     7681 gcaggtgccc tcagcttagg tggccttttc tggaacgtcg ctgtatctgg ccacggcgct
     7741 ttcgtattgt tcgtaaaatt tgccctgctc ggctttcaga atttcaatac ggtggtcgcc
     7801 aaagtggaac tcaggcatat taactgcgtt gctaggtttt ctgctacgat aggtggttct
     7861 cagatgtgag gtcaggtggt tgccgtcggt acacttcagg gccatcagac tctggccaca
     7921 caaaacacca tcacgcggat acagcatttc cacgcagggc tcccagcctg ctgttttttt
     7981 ctgcatcacc ggaccatttg cggggaagtt ggtgcccaga actttcacct tgtagatcag
     8041 acaattacct tccaggctcg tatcctggtg tgcggtcagc acgccgccat cctcgaagat
     8101 ctgtgtacgt tcccaggtaa aaccttccgg aaagctctct ttaaaataat ccggaatacc
     8161 ggaaacgtgc ttgataaagg ttttgctgcc gtacatacag caaggggcca ggatatcgaa
     8221 ggcaaaaggc agaggtcctc cctcggtaac acgaatccgc atattctggg tgccttcaaa
     8281 cggtttacct tctccctcgc ccacacattt gaagtggtag ccgttcacgg ttccttccat
     8341 cgtcatcttg acgcacatat ccttcttcac caggcttgcc atgctgagga cctcctcttt
     8401 ttaattacga cctcgggctc acattatatt gttatccgct cacaatgtca atcatttaac
     8461 acctgcgcca tggtggcggc ttgattactt aactaagatt tacgagggta ggaagtggta
     8521 cggaaagttg gtataagaca aaagtgttgt ggaattgctc caggcgatct gacggttcac
     8581 taaactagct ctgcttttat aggcgcccac cgtacacgcc taaacgtaat acgttctcta
     8641 tcactgatag ggagtaaact ggatatacgt tctctatcac tgatagggag taaactgtag
     8701 atacgttctc tatcactgat agggagtaaa ctggtcttac gttctctatc actgataggg
     8761 agtaaactcc ttatacgttc tctatcactg atagggagta aagtctgcaa acgttctcta
     8821 tcactgatag ggagtaaact cttgatacgt tctctatcac tgatagggag taaactcgag
     8881 tatgtcgagg taggcgtgta cggtgggagg tctatataag cagagcttgt ttagtgaacc
     8941 gtcagatcgc tcgcctattt acgcatgcgc tgagccacca tgggctctta cggcgccctg
     9001 cttttccggg aaaagatccc ctacgtggtg gaaatggaag gcgacgtcga ggggatgaag
     9061 ttcagcgtgc ggggaaaagg ccacggcgac gctaataccg gcaaaatcga ggccagcttt
     9121 atctgcacca caggcgagct gcctgtgcct tggagcagta tcctgacaac cgtgacctac
     9181 ggcgctcagt gtttcgccaa gtacccaaac gatatcaagg actaccccaa gagcgccatg
     9241 cccgagggtt acgtccagga gcggaccatc acctttgaga acgacggagt ttacaagaca
     9301 agagctgaag tgacctacga gaagggctct gtgtataaca gagtgaccct gaacggcagc
     9361 ggcttcaaga agggcggcaa tatcctggga aagaaactgg agttcaacta caacccccac
     9421 tgtatttacg tactgcccga cgtccagaac aacgggatca agtgctacat caacattgtg
     9481 cacgatgtga tcggaggcgg ccagattatc gccgctcacc aacagctgaa cacccccctg
     9541 ggcggcggcc ccgtggatat ccctcactac caccacatcc aggctcacac catactgtca
     9601 aaggacccca aggaaacacg cgatcacatg aacgtggtgg aggtgttcag agctatcgac
     9661 tgcaagaccg cgtacgccgg aagcggcgag ggcagaggca gtctgctgac ttgcggcgat
     9721 gtggaggaaa atcctggacc tatgagcaga ctggacaagt ccaaagtgat taacagcgcc
     9781 cttgaactgc tgaacggcgt tggaatcgaa ggcctgacaa cccggaagct ggcccagaag
     9841 ctgggggtgg agcagcctac actgtactgg cacgtgaaga acaagagagc actcctggac
     9901 gccctgccta tcgagatgct ggacagacac cacacacaca gctgccctct ggaaggcgag
     9961 agctggcagg acttcctgcg gaacaacgcc aagtcttata ggtgcgccct gctgagccac
    10021 cgggacggcg ccaaggtgca cctgggcacc agacctaccg agaaacagta cgagacactg
    10081 gagaaccagc tggccttcct gtgccagcaa ggcttctccc tggagaacgc cctgtacgcc
    10141 ctgagcgccg tgggacactt caccctgggc tgtgtgctgg aagagcagga gcaccaagtg
    10201 gccaaagagg agagggaaac cccaaccacc gactctatgc ctcctctgct gaaacaggct
    10261 atcgaactgt tcgatagaca gggcgccgag cctgcctttc tgttcggcct cgagctgatt
    10321 atctgtggcc tggaaaagca gctgaagtgc gagtctggcg gacccaccga cgctctggac
    10381 gattttgatc tggatatgct gcctgccgac gctctggacg attttgatct ggatatgctg
    10441 ccagccgacg ccctcgacga cttcgacctg gatatgttac ctggctgact agtgaacttg
    10501 tttattgcag cttatattgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
    10561 gcattttttt cactgccaat tgagtcaaaa gcctccggtc ggaggctttt gacttactgt
    10621 tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga
    10681 aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag ttattctttt
    10741 gcacgaggtc tggtgctagg tcttctatta ccgctatctg ccaggacttc cacacaaccg
    10801 tcggtccaaa cggcggcgct tcttctggcg atctgggtac ggcccacggt gcctgcgccg
    10861 ctgcgcacga tcgcgtcgca gcgaccttgg gcccacgcgg cgtcgtcgaa gttgccgtca
    10921 accaggctct gatacagctg atccagtccg atccggagca tgtaggcacg cagtctaggg
    10981 ctacctgcca gttccgggtg acgtctttca aaatagcgtg tctgctgttc catacaggcc
    11041 agccacggcc gccagaagaa aatatttgca acttcgtatt gggagtcgcc gaacatcgcc
    11101 tcgctccaat cgatcacggc ggtaatacgg ccgttgtcgg tcagcacgtt attactacca
    11161 aaatcggcgt gaaccaggtg ccgcacttca ggacaatctt ctgcccacag catcagttcg
    11221 tccagtgcct gagccacgct ggcggaaact gtatcgtcca tcacggtctg ccagtggtac
    11281 acgtgaggat ctgcaatcgc acaaataaaa tcacgccagg tggtgtactg gccgatgccc
    11341 tgaggtccaa acggaccaaa cccgctggtc tggctcagat cagctgcggc aatcgcgtcc
    11401 attgcttcgg caaccggctg cagaacagca ggcagctcgg tttccggcag atcctggagt
    11461 gtaacaccct gtgcacgacg gctgatgcag taggtcagag actcgctgaa ctcgccaata
    11521 tccagaacct cggggatggg cagggctgca cttgcaaagt gacgatacac gtaacgatct
    11581 ttataaaaac cgtcggcgca gctgttcact ctcagcacgt agcctcttcc gccgacgtcg
    11641 aagctaaacg cccggctctc ttcgccttcg ctcagctgca tcagatcgct aacgctatcg
    11701 aacttctcga tcagaaattt ttcaacgctt gtggctgtca gttccggttt cttcatacca
    11761 gaacctcctg gacccggatt ttcctcaacg tcaccacagg tcagcaggct accacgaccc
    11821 tctatattat acgaaccgat caggcttttg tcaaggccct gtgggaggaa gagaagaggt
    11881 caggaattcg ccccactgtg gggtggaggg gacagataaa agtacccaga accagagcca
    11941 cattaaccgg ccctgggaat ataaggtggt cccagctcgg ggacacagga tccctggagg
    12001 cagcaaacat gctgtcctga agtggacata ggggcccggg ttggaggaag aagactagct
    12061 gagctctcgg acccctggaa gatgccatga cagggggctg gaagagctag cacagactag
    12121 agaggtaagg ggggtagggg agctgcccaa atgaaaggag tgagaggtga cccgaatcca
    12181 caggagaacg gggtgtccag gcaaagaaag caagaggatg gagaggtggc taaagccagg
    12241 gagacggggt actttggggt tgtccagaaa aacggtgatg atgcaggcct acaagaaggg
    12301 gaggcgggac gcaagggaga catccgtcgg agaaggccat cctaagaaac gagagatggc
    12361 acaggcccca gaaggagaag gaaaagggaa cccagcgagt gaagacggca tggggttggg
    12421 tgagggagga gagatgcccg gagaggaccc agacacgggg aggatccgct cagaggacat
    12481 cacgtggtgc agcgccgaga aggaagtgct ccggaaagag catccttggg cagcaacaca
    12541 gcagagagca aggggaagag ggagtggagg aagacggaac ctgaaggagg cggcagggaa
    12601 ggatctgggc cagccgtaga ggtgacccag gccacaagct gcagacagaa agcggcacag
    12661 gcccagggga gagaatgctg gtcagagaaa gcacgtaagg taccgcggtc tagattgatc
    12721 agcctcgact ctagcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc
    12781 tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagct
    12841 cagatcattg tcacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    12901 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    12961 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    13021 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    13081 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    13141 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    13201 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    13261 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
    13321 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
    13381 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
    13441 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
    13501 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    13561 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    13621 aatgaagttt taaatcaagc ccaatctgaa taatgttaca accaattaac caattctgat
    13681 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
    13741 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
    13801 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
    13861 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
    13921 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
    13981 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
    14041 gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
    14101 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
    14161 tcttctaata cctggaatgc tgtttttccg gggatcgcag tggtgagtaa ccatgcatca
    14221 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
    14281 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
    14341 aactctggcg catcgggctt cccatacaag cgatagattg tcgcacctga ttgcccgaca
    14401 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
    14461 ctcgacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
    14521 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
    14581 tgagacacgg gccagagctg ca
//
