LOCUS       sequence-271856-        8698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of gRNA i2 targeting TCF4 for CRISPRi; U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8698)
  AUTHORS   Hazelbaker DZ, Beccard A, Angelini G, Mazzucato P, Messana A, Lam D,
            Eggan K, Barrett LE
  TITLE     A multiplexed gRNA piggyBac transposon system facilitates efficient 
            induction of CRISPRi and CRISPRa in human pluripotent stem cells.
  JOURNAL   Sci Rep. 2020 Jan 20;10(1):635. doi: 10.1038/s41598-020-57500-1.
  PUBMED    31959800
REFERENCE   2  (bases 1 to 8698)
  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: "Sci Rep."; 
            date: "2020-01-20"; volume: "10(1):635. doi"; pages: " 
            10.1038/s41598-020-57500-1"
COMMENT     Sequence Label: pPN434
FEATURES             Location/Qualifiers
     source          1..8698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          571..798
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      810..819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             819..884
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             891..911
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             936..5036
                     /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             936..5036
                     /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             5037..5084
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5112..5129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5118..5325
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5334..5474
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5334..5463
                     /label=AAV2 ITR
     rep_origin      5549..6004
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5636..5655)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5846..5867
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6021..6040)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6140..6162
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6200..6218)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             6625..7416
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      7599..8187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8088..8107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8265..8513
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8265..8285
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8436..8455
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8535..8610
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
ORIGIN
        1 tgagacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg
      421 ccaggcgggg cggggsgggg sgrggggsgg ggsggggsgr ggsggrgagg kgcggcggca
      481 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
      541 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
      601 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
      661 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagctgag caagaggtaa
      721 gggtttaagg gatggttggt tggtggggta ttaatgttta attacctgga gcacctgcct
      781 gaaatcactt tttttcaggt tggaccggtg ccaccatgga ctataaggac cacgacggag
      841 actacaagga tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga
      901 agcggaaggt cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg
      961 acatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca
     1021 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag
     1081 ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa
     1141 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1201 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1261 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1321 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1381 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1441 tcgagggcga cctgaacccc gacaacagcg acgttgacaa gctgttcatc cagctggtgc
     1501 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1561 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     1621 ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc
     1681 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     1741 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     1801 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     1861 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     1921 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     1981 ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg
     2041 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2101 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2161 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2221 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2281 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2341 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga
     2401 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2461 agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg
     2521 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg
     2581 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     2641 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     2701 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     2761 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     2821 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     2881 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     2941 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     3001 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3061 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3121 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3181 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3241 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3301 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3361 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3421 tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg
     3481 actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg
     3541 tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca
     3601 agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     3661 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     3721 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga
     3781 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     3841 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     3901 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     3961 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     4021 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4081 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg
     4141 gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc
     4201 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4261 tcctgcccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga
     4321 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4381 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4441 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4501 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4561 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     4621 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     4681 aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca
     4741 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag
     4801 tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca
     4861 tccacctgtt taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca
     4921 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     4981 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa
     5041 ggccggcggc cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcctagagc
     5101 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5161 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5221 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5281 cagcaagggg gaggattggg aagagaatag caggcatgct ggggagcggc cgcaggaacc
     5341 cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg
     5401 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
     5461 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     5521 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
     5581 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
     5641 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5701 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5761 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5821 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5881 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
     5941 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
     6001 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
     6061 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     6121 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     6181 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     6241 ataatggttt cttagacgtc aggtggcact tccagccagg acagaaatgc ctcgacttcg
     6301 ctgctaccca aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag
     6361 tggacataag cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac
     6421 tggtccagaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt tttcatggct
     6481 tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt
     6541 tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt
     6601 cgccctaaaa caaagttaaa cattatgagg gaagcggtga tcgccgaagt atcgactcaa
     6661 ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat
     6721 ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga tttgctggtt
     6781 acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa
     6841 acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg
     6901 cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
     6961 cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct
     7021 atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa
     7081 ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta
     7141 tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
     7201 atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca
     7261 atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt
     7321 ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccactac
     7381 gtgaaaggcg agatcaccaa ggtagtcggc aaataaaagc attggtaact gtcagaccaa
     7441 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     7501 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7561 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7621 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7681 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7741 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7801 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7861 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7921 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7981 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     8041 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     8101 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     8161 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     8221 gccttttgct ggccttttgc tcaggtctca ctctagaggc atgtgagggc ctatttccca
     8281 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
     8341 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
     8401 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
     8461 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac accgcatcac
     8521 catggactcc cccggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     8581 caacttgaaa aagtggcacc gagtcggtgc ttttttgttt tagagctaga aatagcaagt
     8641 taaaataagg ctagtccgtt tttagcgcgt gcgccaattc tgcagacaaa tggctcta
//
