LOCUS Exported 12490 bp ds-DNA linear SYN 20-MAY-2021 DEFINITION Donor construct for introduction of hNIL factors and SBP-LNGFR for streptavidin bead enrichment. ACCESSION . VERSION . KEYWORDS CLYBL-(Ef1a-SBP-LNGFR-T2A-mApple)-(CAG-rtTA)-(TRE-hNIL)) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12490) AUTHORS Fernandopulle MS, Prestil R, Grunseich C, Wang C, Gan L, Ward ME TITLE Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. JOURNAL Curr Protoc Cell Biol. 2018 Jun;79(1):e51. doi: 10.1002/cpcb.51. Epub 2018 May 18. PUBMED 29924488 REFERENCE 2 (bases 1 to 12490) AUTHORS . TITLE Direct Submission JOURNAL Exported May 20, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12490 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 8..27 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" CDS 59..805 /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" primer_bind complement(896..915) /label=Bglob-pA-R /note="Rabbit beta-globin polyA region, reverse primer" polyA_signal 961..1016 /label=-beta-globin poly(A) signal /note="rabbit beta-globin polyadenylation signal (Gil and Proudfoot, 1987)" primer_bind complement(1015..1034) /label=rbglobpA-R /note="Rabbit beta-globin polyA, reverse primer. Also called rb-glob-pA-term-R" primer_bind 1326..1343 /label=L4440 /note="L4440 vector, forward primer" protein_bind 1460..1481 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 1496..1526 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1534..1550 /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)." primer_bind 1539..1561 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1558..1574 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1558..1574 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" polyA_signal 1623..1744 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1634..1653) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 1688..1707 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(2976..3029) /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" regulatory 3301..3310 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(4101..4157) /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" primer_bind complement(5077..5101) /label=LNCX /note="Human CMV promoter, forward primer" protein_bind 5146..5164 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5182..5200 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5218..5236 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5254..5272 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5290..5308 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5326..5344 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 5362..5380 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" rep_origin complement(6406..6994) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6486..6505) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7165..8025) /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 7788..7807 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" protein_bind 8883..8916 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 8917..8942 /label=SA /note="splice acceptor site" CDS 8966..9019 /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 9029..9628 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(9029..9048) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 9525..9545 /label=Puro-F /note="Puromycin resistance gene, forward primer" polyA_signal 9649..9873 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 9909..10120 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 10133..10401 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" regulatory 10439..10448 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 10526..10639 /codon_start=1 /product="streptavidin-binding peptide" /label=SBP /note="selected from a peptide library; binds streptavidin with nanomolar affinity (Keefe et al., 2001)" /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP" CDS 11423..11476 /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" regulatory 11483..11492 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" protein_bind 12206..12239 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(12255..12272) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 12261..12485 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" ORIGIN 1 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga atgtcgacat 61 gagccggctg gacaagagca aagtgatcaa cagcgccctg gaactgctga acggcgtggg 121 catcgagggc ctgaccaccc ggaagctggc ccagaaactg ggcgtggaac agcccaccct 181 gtactggcac gtgaagaaca agcgggccct gctggacgcc ctgcccatcg agatgctgga 241 ccggcaccac acccacagct gccctctgga aggcgagagc tggcaggact tcctgcggaa 301 caacgccaag agctacagat gcgccctgct gagccaccgg gacggcgcca aagtgcacct 361 gggcaccaga cccaccgaga agcagtacga gacactggaa aaccagctgg ccttcctgtg 421 ccagcagggc ttcagcctgg aaaacgccct gtacgccctg agcgccgtgg gccacttcac 481 cctgggctgc gtgctggaag aacaggaaca ccaggtcgcc aaagaggaaa gagagacacc 541 caccaccgac agcatgcccc ccctgctgaa gcaggccatc gagctgttcg acagacaggg 601 cgccgagccc gccttcctgt tcggcctgga actgatcatc tgcggcctcg agaagcagct 661 gaagtgcgag agcggcggac ccaccgacgc cctggacgac ttcgacctgg acatgctgcc 721 cgccgatgcc ctggatgatt ttgatctgga tatgctccct gctgacgctc tcgatgactt 781 cgatctcgat atgctgccag gctaaaacgc gtgaattcac tcctcaggtg caggctgcct 841 atcagaaggt ggtgactggt gtggccaatg ccctggctca caaataccac tgagatcttt 901 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct 961 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg 1021 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt 1081 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc 1141 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag 1201 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt 1261 ccttacatgt gcggccgtcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg 1321 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc 1381 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg 1441 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac 1501 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag 1561 gaaacagcta tgaccatgat tacgccaagc tctagctaga ggtcgacggt atacagacat 1621 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 1681 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 1741 agttggggtg ggcgatcgat tcagaactgg gcgtgatcca cttcgtccag ccaggaggca 1801 gggctggcag gaaaatcggg gtagccgcca gagctgcctg tgctcagatc gctgctaggg 1861 ccattgccgg ccagcactct cattggagga ggtccgcctg gagcgccaga tggcaccagg 1921 cccaggcttg tatcggggta caccaggctg gacagcagag gctgaggtcc tggcagagac 1981 tgaggggcag ctggagaagg tggcacgccg taagggctgc cgggcctcag ttctctatac 2041 tgctcgggtc cggccagtcc gccgtgttcc agagagaagt tgcccagggc gccagaaggt 2101 ctgcccagag cctgggttgg ctctcccagg ctgccataca ggccattggc tgggcccatt 2161 tcggccaggc taggctcatc tgggaaggac acctcggcgt cgctgtcctg gccttcctgc 2221 acgctgtcct tgtcgctctt gctgccgcct ctggaccgct tcatgttccg gaagtactgg 2281 ccccatctct gcctgccggc gtccttcttc agccgcttct ctttggcccg tctgttctgg 2341 aaccacactt gcaccactct catgtccagg cctgtctcgc tgctcagctg ttcccgcaca 2401 tgtctggcgg gcttggggct ggtgttgtag gcggacttca gggtttccag ctgcttggcg 2461 gtgatggtgg ttctgggtct tttggcggtg gcctcggcct ctctctgctt ggctgtctcg 2521 tagtcggctt tacacacgag ccggctatct tccatcaggt agaactcgtc gccggtggcc 2581 agctgtctct tgcacacgac gcaggcgaag cagtgcagat ggtacacgaa gtcctgggct 2641 ctccgcacga cctgtgtagg aggaattccc agctggcagg cggcgcactt ggtgccgaat 2701 ctcttgaaga agtcgtcctt gcagtacacg ctctcgcctc tgctaaagca tctctcggcc 2761 aggggggtgt ggcagtcgct gcacttcagg cacttgctgt gccagtgtct gtccagggcc 2821 ttcaggataa accggtccag gatgtgctgg tcgcagccgg cgcacagggg gatttctctt 2881 ctcaggtcgg cccttctagc cagcagggcc agcagcagat ctccgccggc agattctctg 2941 gcagggccca gttcgcctct ggcttccatg gtaccggggc cagggttctc ttccacgtcg 3001 ccacatgtca gcaggctgcc tctgccctcg ccagagcctc tgctgtgcat caatgcctca 3061 ataggactgg ctaccatgct gttaggtgta tctggaagtt gagaggacat tgatgctact 3121 tcactgccag tggaattaga gcccggtcct ccttctgaaa aattgaccag ttgctgaaaa 3181 gcaggctgat ctatgtcact ctgcaaggcg aagtcgctca gtactttcca aggtggctgg 3241 taactttgta cttccactgg gttagcctgt aagccaccgt cgtgtctctc tggactggca 3301 gccaccatgg gagttcctgt catcccctgg atattagttt tgtcattggg ctgctgctgc 3361 tggagttgct tcatcatgat gcttcgcttc ttgtccttgc accgcttgtt ttgaaaccag 3421 acccggatca cacggggact gaggcccgtc atctctacca gttgctcctt catgagcgca 3481 tctggccgcg ggtttgcggc gtagcaggtc cgcaaggtgt gcagctgctt ctcgttcagc 3541 acagtccgca cgcgggtggt cttctccggc tgcttgtgga cgtggggccg cagggccggc 3601 tgcctggcgg agatgggctc cgctgccatt tgcagtggcc gcgctggatg caggggactg 3661 agcgggtcgc cagcgcctag actggccctc tccaccacat cgtggtctgc tcggcagaag 3721 agaccgtcct cccgaagcgc aaattcgtcc ccagggatga gctggcggct gcaggccaca 3781 cagcggaaac actcgatgtg atacaccttg gagcgggcac gcatcacgaa gtcgttcttg 3841 ctgaagccga tgctgcactt ggcgcatttg atcccgtaca acctgatata atctctttta 3901 cagtaggttt tcccatccct aacaaagcat gtacagctct cgtccaaata ctgattacac 3961 tccgcacatt tcaaacatgc cgcatgccat tccaaatccg gagaaaccct cagaatatac 4021 tgatcgtgaa tctgattgcc gcaaccaaca catagggaaa tcagacgttt tttttttggt 4081 ggatctccca tgtctcccat aggtccaggg ttctcctcca cgtctccagc ctgcttcagc 4141 aggctgaagt tagtagctcc ggatcccaag atacaatccc tggctatggg gaggtgaggt 4201 gcatagcggt gcttgtcggg aggtgggggc tgccaatagt ccatgtctga cccggccggg 4261 ctggcgggcg ataaagtgca gctgtagggg gaggtggaat tggaggacac ggaggaggac 4321 ggcgcggggc tgttggtgca actccacgtg gaggcgggcg aggggctgtc tccgctgctg 4381 ctcagggcgg cgctggcgcc tcccgggctc agcaacactg cctcggagaa gagcgccccc 4441 ggcaggcccc cgccgccgcc cccgcagtga tccgccaggc gcagggtctc ggtgagtgcc 4501 cagatgtagt tgtgggcgaa gcgcagggtc tcgatcttgg tgagcttggc gtcctcgggg 4561 aacgtgggga gcacctcgcg cagcgcgtcc agtgccgcgt tgaggttgtg catgcggttt 4621 cgctcgcggt tgttggcctt cagtctacgg gtcttcttga tgcgctgcac cgtctcggcc 4681 gtcttggcgc ctcgggagac ggcccgcgcc cgggaagggc gccgtttgca atcgtgtacc 4741 agacccagca gccgtgcggg ccggcagccc tccgcacccg cagccacgcc gccccgcgcc 4801 ccctgcccgg cctcagcccc gcgctgccga cgcgccccgc ctgacgcgcc cggctcctcc 4861 tcctcttctt cgtcggcgct ggatgacagc ggggtcaggg ccgccaaggc gggggaggcc 4921 gatccgagca gcactaacac gtcctcttcc tccttcaact ccaaggtctc ggatttgacg 4981 aacatggtgg cggcttaatt aaccttaagt ttacgagggt aggaagtggt acggaaagtt 5041 ggtataagac aaaagtgttg tggaattgct ccaggcgatc tgacggttca ctaaacgagc 5101 tctgctttta taggcgccca ccgtacacgc ctaaagctta tacgttctct atcactgata 5161 gggagtaaac tggatatacg ttctctatca ctgataggga gtaaactgta gatacgttct 5221 ctatcactga tagggagtaa actggtcata cgttctctat cactgatagg gagtaaactc 5281 cttatacgtt ctctatcact gatagggagt aaagtctgca tacgttctct atcactgata 5341 gggagtaaac tcttcatacg ttctctatca ctgataggga gtaaaccact gtggggtgga 5401 gatatctcct catccttcca acataaatat attttgggat tatatcaaca ttcaatgtta 5461 cttaaagtga ccttgtaaat attttcacaa ctgagccatg tttgatttgt atacttatgt 5521 ttactttact gtttttcctg aagttaataa ttgccttgaa tttatttatt tctttaaaaa 5581 tgtttcatta ctcaggactg tagtttacat tacgattctt tgtgttatac agttgatggg 5641 tttcttttct ttcttaattt ctttaaaaaa tagagatggg gtcttactat attacccagg 5701 ctggtcttga agtcctgggc tcaagtgatc ttcctgtctc agcctaccaa gtagctgaga 5761 ctataggtgc aaaaaagcca ctatacctgg ctagtttaca ggttttaaca aatgcattat 5821 gccacgtatc cattattaca ggatcacaca agatattttc attaccctga aagcatccct 5881 gtgttccacc aattcatcct gcctccatga gccgctggca accactgatc tctatagttt 5941 tgccttttct aaaatgtcat ataattggaa tcatacagtc tgtagcattt tcagactagc 6001 ttttaaaatt tggcaatatg catttaaggt tcctccttaa atgtgaaggg catagccaat 6061 gtggcttgat agctcatttc tttttattgg tgaatatttc attgtctgga tgttccacag 6121 tttgtttatc cattcaccta ttcaatttgc tttttttctg tgtatctatc actaattcaa 6181 tactggactc tccaacagag ccgtcccgct tccctaaggc cctgctctgg gcttctgggt 6241 ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct tcctcgtcca 6301 ccatctcatg cccctggctc tcctgcccct tccctacagg ggttcctggc tctgctctaa 6361 gggcgaattc gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6421 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6481 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6541 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6601 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6661 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6721 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6781 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6841 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6901 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6961 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 7021 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 7081 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 7141 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 7201 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7261 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 7321 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7381 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7441 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 7501 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7561 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7621 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7681 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7741 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7801 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7861 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7921 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7981 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 8041 atagcaggag aatcatgcca atgggccaat atacattctg acccacagtt tcataataaa 8101 ataaaatggt tgtggttgta agccactatg tttcagagtg gtttgttaca cagcaataaa 8161 taactaatat agtaggcata ccatcaagtc caaagtaggt agagaagaat gtaaatagca 8221 gagcaaaaca gcatgactgg tggctgggag gcttaaaact gggacaggat cagagtcatg 8281 aaagaagtca aagaaatggt tcagaagtaa ggctgagact gacttacaaa agctgaaagt 8341 ccctttaagt tggtgtttgg tgcattggca ggggcaggta tggtgactta aaagagccat 8401 gctcaacaag atcaagcaca acacaatcac gggtcacccc agcagacctt agcgagtcta 8461 gccatttctt tggtggtggt cacagtcatg cttcagccca gtttccactt ggacaaatgg 8521 tacatatttt caatgagatg aaaattaaga tacaatccat gtgctcagag agtgatcaca 8581 gctctgacta aacactgtgc cccaaagtgt tgaggaattg ggaaaaccta gctgagttag 8641 tggtctcttt tctgttacaa taaagctcat aatgaaaatt agccttcttt gttcttcccc 8701 aagtctttct ttctagacga aactactttc aactgtttta acttccttac tgttaacttc 8761 catatttcta gatagtatgg tcagactgtt atttcttgac ttttaaatgt aagatattat 8821 ctactgactt ccttctatgt aagatgagga ttgagctctc ttacccttct cccagcggcc 8881 gcataacttc gtatagcata cattatacga agttatctga cctcttctct tcctcccaca 8941 gggcctcgag agatctggca gcggagaggg cagaggaagt cttctaacat gcggtgacgt 9001 ggaggagaat cccggcccta ggctcgagat gaccgagtac aagcccacgg tgcgcctcgc 9061 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 9121 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga 9181 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 9241 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 9301 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 9361 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 9421 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 9481 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta 9541 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 9601 gtgcatgacc cgcaagcccg gtgcctgatc tagagggccc gtttaaacct gtgccttcta 9661 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 9721 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 9781 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 9841 gcaggcatgc tggggatgcg gtgggctcta tggatgcatg atctgcgatc gctccggtgc 9901 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 9961 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 10021 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 10081 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 10141 catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 10201 cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 10261 aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 10321 ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 10381 tgttctgcgc cgttacagat ccaagctgtg accggcgcct acgctagcgc taccggtcgc 10441 caccatgggc tggtcatgta tcattctgtt tctggtcgca accgcaactg gagtgcattc 10501 acaggtgcag ctcgaggggt cagggatgga cgaaaagacc acaggatggc gaggaggaca 10561 cgtggtcgag ggactggcag gagagctgga acagctgcgg gctagactgg aacaccatcc 10621 tcagggacag cgagagccag gaagtggagc gatcgcgaag gaagcctgcc ccacagggct 10681 gtacactcat tctggagaat gctgtaaagc ttgtaacctg ggagagggag tggcacagcc 10741 atgcggagcc aatcagactg tgtgcgagcc ttgtctggac tccgtcacat tctctgatgt 10801 ggtcagtgcc acagaacctt gcaagccatg tactgagtgc gtgggcctgc agtctatgag 10861 tgctccttgt gtggaggctg acgatgcagt ctgccggtgt gcatacggat actatcagga 10921 cgagactacc ggcagatgtg aagcttgcag ggtgtgtgag gcaggctcag ggctggtctt 10981 tagctgccag gataaacaga acaccgtgtg cgaggaatgt cctgacggga catatagcga 11041 tgaggccaat cacgtggacc cctgcctgcc ttgtactgtg tgcgaggata ccgaaaggca 11101 gctgcgcgaa tgtaccagat gggcagacgc cgagtgcgag gaaatcccag ggcgatggat 11161 tactcggtcc accccccctg aaggatcaga cagcaccgca ccatctacac aggagccaga 11221 agcaccacca gagcaggatc tgatcgcctc caccgtggct ggcgtggtca caactgtcat 11281 ggggagctcc cagccagtgg tcacccgggg caccacagat aacctgattc ccgtgtattg 11341 ctccatcctg gcagccgtcg tcgtcggact ggtggcatac atcgccttca agcggcaatt 11401 gatgcacagc agaggctctg gcgagggcag aggaagtctt ctaacatgcg gtgacgtgga 11461 ggagaatccc ggcccttcaa acgccaccat ggtgtccaag ggcgaagaga acaacatggc 11521 catcatcaaa gaattcatgc ggttcaaggt gcacatggaa ggcagcgtga acggccacga 11581 gttcgagatt gaaggcgaag gcgagggcag accctacgag gcttttcaga ccgccaagct 11641 gaaagtgacc aaaggcggcc ctctgccttt tgcctgggac attctgagcc ctcagtttat 11701 gtacggcagc aaggtgtaca tcaagcaccc cgccgacatt cccgactact tcaagctgag 11761 cttccccgag ggcttcagat gggagagagt gatgaacttc gaggacggcg gcatcatcca 11821 cgtgaaccag gatagctctc tgcaggacgg cgtgttcatc tacaaagtga agctgcgggg 11881 caccaacttt ccctctgatg gccccgtgat gcagaaaaag accatgggct gggaagccag 11941 cgaggaaaga atgtaccctg aggatggcgc cctgaagtcc gagatcaaga agcggctgaa 12001 gctgaaggat ggcggccact atgccgccga agtgaaaacc acctacaagg ccaagaaacc 12061 cgtgcagctg cctggcgcct acatcgtgga tatcaagctg gacatcgtgt cccacaacga 12121 ggactacacc atcgtggaac agtacgagag agccgaaggc agacacagca caggcggaat 12181 ggacgagctg tacaagtaag gtaccataac ttcgtatagc atacattata cgaagttata 12241 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 12301 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 12361 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 12421 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 12481 tatgggtctc //