LOCUS       sequence-422949-       10424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express a single-guide RNA under a U6 promoter, accompanied by 
            the expression of eCas9 and the affinity sorting tag 
            TST-EGFP-GPIBY55 under separate promoters..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10424)
  AUTHORS   Yang L, Cui L, Ma S, Zuo Q, Huang Q
  TITLE     A Gene Transfer-Positive Cell Sorting System Utilizing 
            Membrane-Anchoring Affinity Tag.
  JOURNAL   Front Bioeng Biotechnol. 2022 Jun 16;10:930966. doi: 
            10.3389/fbioe.2022.930966. eCollection 2022.
  PUBMED    35782508
REFERENCE   2  (bases 1 to 10424)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-06-16"; volume: "10:930966. doi"; 
            pages: " 10.3389/fbioe.2022.930966. eCollection 2022"
COMMENT     Sequence Label: pCRISPR/eCas9-GPIBY55
FEATURES             Location/Qualifiers
     source          1..10424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          984..1211
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1223..1232
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1232..1297
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1304..1324
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1349..5449
                     /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
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5450..5497
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5525..5542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5531..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        5802..6105
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6106..6309
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6260..6280
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(6425..6430,6431..6454,6455..6490,6491..6514)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     regulatory      6521..6530
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6527..6529,6530..6532,6533..7243)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6527..6529,6530..6532,6533..7243)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6833..6852)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7180..7201
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    7373..7429
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    7553..7674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7590..7609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7644..7663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7687..7827
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7687..7816
                     /label=AAV2 ITR
     rep_origin      7902..8357
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7989..8008)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8199..8220
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8374..8393)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8493..8515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8553..8571)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8639..8743
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8744..8812,8813..9604)
                     /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(8962..8981)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9775..10363
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10264..10283
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggsg gggcgrgggg gggggggggg sgrggsggrg rggkgsggsg gcrgccaatc
      901 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
      961 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     1021 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     1081 gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta
     1141 agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca
     1201 ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa
     1261 ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa
     1321 ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg
     1381 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     1441 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     1501 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     1561 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     1621 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     1681 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     1741 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     1801 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     1861 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     1921 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     1981 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     2041 gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt
     2101 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     2161 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     2221 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     2281 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     2341 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     2401 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     2461 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     2521 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     2581 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     2641 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     2701 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     2761 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     2821 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     2881 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     2941 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     3001 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     3061 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     3121 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     3181 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     3241 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     3301 gcggcggaga tacaccggct ggggcgcgct gagccggaag ctgatcaacg gcatccggga
     3361 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     3421 cttcatggcg ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     3481 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     3541 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     3601 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     3661 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     3721 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     3781 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     3841 gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat
     3901 cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc
     3961 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     4021 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     4081 taaggccggc ttcatcaaga gacagctggt ggaaacccgg gcgatcacaa agcacgtggc
     4141 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     4201 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     4261 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     4321 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     4381 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     4441 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     4501 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     4561 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     4621 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     4681 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     4741 cggcttcgat agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     4801 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     4861 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     4921 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     4981 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     5041 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     5101 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     5161 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     5221 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     5281 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     5341 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     5401 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc
     5461 ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga
     5521 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     5581 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     5641 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     5701 ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccttatta atagtaatca
     5761 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     5821 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     5881 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     5941 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     6001 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     6061 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     6121 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     6181 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     6241 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     6301 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggtacca tgctgctgga
     6361 gcctggcaga ggctgctgcg ctctggccat cctgctggcc atcgtggaca tccagagcgg
     6421 aggcagcgcc tggagccacc ctcagttcga gaaaggagga ggcagcggcg gcggaagcgg
     6481 aggctccgct tggtcccacc cacagttcga aaagccggtc gccaccatgg tgagcaaggg
     6541 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     6601 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     6661 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     6721 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     6781 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     6841 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     6901 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     6961 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     7021 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     7081 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     7141 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     7201 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtccggcc ggactcagat
     7261 ccggcagcac ctggagttca gccacaacga gggcaccctg agcagcggct tcctccagga
     7321 gaaggtgtgg gtgatgctgg tgaccagcct ggtggctctc caggccctgt agagatctcg
     7381 agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccgggatcca ccggatctag
     7441 ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     7501 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     7561 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     7621 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt
     7681 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
     7741 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
     7801 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
     7861 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     7921 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt
     7981 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     8041 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     8101 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     8161 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     8221 aacaacactc aactctatct cgggctattc ttttgattta taagggattt tgccgatttc
     8281 ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     8341 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     8401 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     8461 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     8521 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     8581 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     8641 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     8701 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     8761 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     8821 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     8881 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     8941 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     9001 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     9061 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     9121 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     9181 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     9241 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     9301 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     9361 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     9421 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     9481 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     9541 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     9601 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     9661 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     9721 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     9781 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     9841 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     9901 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     9961 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
    10021 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
    10081 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
    10141 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
    10201 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
    10261 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
    10321 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
    10381 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
