LOCUS       sequence-251390-       13382 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CRISPR-mediated repression of ZFAND3. KRAB domain and Catalytically 
            inactive Cas9 from S. pyogenes with 2A-BlastR under the hPGK 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13382)
  AUTHORS   Miguel-Escalada I, Bonas-Guarch S, Cebola I, Ponsa-Cobas J, 
            Mendieta-Esteban J, Atla G, Javierre BM, Rolando DMY, Farabella I, 
            Morgan CC, Garcia-Hurtado J, Beucher A, Moran I, Pasquali L, 
            Ramos-Rodriguez M, Appel EVR, Linneberg A, Gjesing AP, Witte DR, 
            Pedersen O, Grarup N, Ravassard P, Torrents D, Mercader JM, Piemonti
            L, Berney T, de Koning EJP, Kerr-Conte J, Pattou F, Fedko IO, Groop 
            L, Prokopenko I, Hansen T, Marti-Renom MA, Fraser P, Ferrer J
  TITLE     Human pancreatic islet three-dimensional chromatin architecture 
            provides insights into the genetics of type 2 diabetes.
  JOURNAL   Nat Genet. 2019 Jun 28. pii: 10.1038/s41588-019-0457-0. doi: 
            10.1038/s41588-019-0457-0.
  PUBMED    31253982
REFERENCE   2  (bases 1 to 13382)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Genet. 
            2019 Jun 28. pii: 10.1038/s41588-019-0457-0. doi: 
            10.1038/s41588-019-0457-0."
COMMENT     Sequence Label: Lenti-(BB)-hPGK-KRAB-dCas9-P2A-BlastR ZFAND3-g...
FEATURES             Location/Qualifiers
     source          1..13382
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        1..76
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        107..617
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     536..554
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      630..639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             666..851
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             873..4976
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTSLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4980..5006
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4986..5012
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             5025..5045
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5052..5072
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5118..5174
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5175..5573
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    5598..6186
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5651..5671)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6069..6080)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6258..6491
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6517..6534)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6523..6747
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6793..7221
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6880..6899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7090..7111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7230..7250)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7235..7564
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7415..7550
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7477..7496
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        7612..7659
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             7678..8052
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    8182..8303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8219..8238)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8273..8292
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(8352..8368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8352..8368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(8365..8387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    8376..8392
                     /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)."
     promoter        complement(join(8400..8406,8407..8424,8425..8430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8445..8466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8583..8600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8754..9342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8834..8853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9513..10304,10305..10373))
                     /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     10136..10155
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10374..10478)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(10553..10572)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        10744..11123
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        11125..11323
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     11278..11298
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             11341..11521
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    11568..11693
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    12186..12419
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             12604..12648
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    12946..13063
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        13114..13362
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     13114..13134
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     13285..13304
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
       61 ggcaccgagt cggtgctttt ttgaattcgc tagctaggtc ttgaaagggg ttggggttgc
      121 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
      181 ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca
      241 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
      301 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
      361 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
      421 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
      481 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
      541 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca
      601 ccgacctctc tccccaggac cggtgatccg ccaccatgga tgctaagtca ctaactgcct
      661 ggtcccggac actggtgacc ttcaaggatg tatttgtgga cttcaccagg gaggagtgga
      721 agctgctgga cactgctcag cagatcgtgt acagaaatgt gatgctggag aactataaga
      781 acctggtttc cttgggttat cagcttacta agccagatgt gatcctccgg ttggagaagg
      841 gagaagagcc cggcggttcc ggcggagggt cgatggacaa gaagtacagc atcggcctgg
      901 ccatcggcac caactctgtc ggctgggccg tgatcaccga cgagtacaag gtgcccagca
      961 agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggcg
     1021 ccctgctgtt cgacagcgga gaaacagccg aggccacccg gctgaagaga accgccagaa
     1081 gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga
     1141 tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg
     1201 ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg
     1261 agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg
     1321 acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga
     1381 tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc
     1441 agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg
     1501 ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc
     1561 ccggcgagaa gaagaatggc ctgttcggca acctgattgc cctgagcctg ggcctgaccc
     1621 ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca
     1681 cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt
     1741 ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca
     1801 ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc
     1861 aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga
     1921 ttttcttcga ccagagcaag aacggctacg ccggctacat cgatggcgga gccagccagg
     1981 aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc
     2041 tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca
     2101 tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt
     2161 acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct
     2221 actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg
     2281 aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgcc agcgcccaga
     2341 gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca
     2401 agcacagcct gctgtacgag tacttcaccg tgtacaacga gctgaccaaa gtgaaatacg
     2461 tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaaa gccatcgtgg
     2521 acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca
     2581 agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct
     2641 ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg
     2701 aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag
     2761 agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc
     2821 agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca
     2881 tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca
     2941 acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga
     3001 aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca
     3061 gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag
     3121 tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca
     3181 cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag
     3241 agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga
     3301 agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca
     3361 tcaaccggct gtccgactac gatgtggacg ctatcgtgcc tcagagcttt ctgaaggacg
     3421 actccatcga taacaaagtg ctgactcgga gcgacaagaa ccggggcaag agcgacaacg
     3481 tgccctccga agaggtcgtg aagaagatga agaactactg gcgccagctg ctgaatgcca
     3541 agctgattac ccagaggaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg
     3601 aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc
     3661 acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaac gacaaactga
     3721 tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt
     3781 tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga
     3841 acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt
     3901 acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg
     3961 gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga
     4021 ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaacag
     4081 gcgagatcgt gtgggataag ggccgggact ttgccaccgt gcggaaagtg ctgtctatgc
     4141 cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta
     4201 tcctgcccaa gaggaacagc gacaagctga tcgccagaaa gaaggactgg gaccctaaga
     4261 agtacggcgg cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg
     4321 aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg
     4381 aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag
     4441 tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc
     4501 ggaagagaat gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct
     4561 ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg
     4621 aggataatga gcagaaacag ctgtttgtgg aacagcacaa acactacctg gacgagatca
     4681 tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaagg
     4741 tgctgagcgc ctacaacaag cacagagaca agcctatcag agagcaggcc gagaatatca
     4801 tccacctgtt taccctgacc agtctgggag cccctgccgc cttcaagtac tttgacacca
     4861 ccatcgaccg gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc
     4921 agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacgcct
     4981 atccctatga cgtgcccgat tatgccagcc tgggcagcgg ctcccccaag aaaaaacgca
     5041 aggtggaaga tcctaagaaa aagcggaaag tggacggcat tggtagtggg agcaacggca
     5101 gcagcgggga tccggccgca acaaacttct ctctgctgaa acaagccgga gatgtcgaag
     5161 agaatcctgg accgatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag
     5221 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc
     5281 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt
     5341 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta
     5401 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg
     5461 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga
     5521 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagaattcg
     5581 atatcaagct tatcggtaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     5641 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5701 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5761 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5821 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5881 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5941 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     6001 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     6061 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     6121 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     6181 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     6241 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
     6301 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     6361 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     6421 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     6481 aatctctagc agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     6541 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6601 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6661 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     6721 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     6781 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6841 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     6901 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6961 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     7021 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     7081 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     7141 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     7201 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     7261 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     7321 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     7381 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     7441 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     7501 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     7561 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     7621 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     7681 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
     7741 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
     7801 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
     7861 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
     7921 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
     7981 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
     8041 gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     8101 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     8161 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     8221 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     8281 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     8341 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     8401 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     8461 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     8521 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     8581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     8641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     8701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     8761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     8821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     8881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     8941 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     9001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     9061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     9121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     9181 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     9241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     9301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     9361 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     9421 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     9481 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     9541 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     9601 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     9661 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     9721 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     9781 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     9841 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     9901 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     9961 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
    10021 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
    10081 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
    10141 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
    10201 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
    10261 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
    10321 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
    10381 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
    10441 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
    10501 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
    10561 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
    10621 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
    10681 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
    10741 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
    10801 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
    10861 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
    10921 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
    10981 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
    11041 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
    11101 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
    11161 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
    11221 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
    11281 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagcgcgttt tgcctgtact
    11341 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
    11401 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
    11461 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
    11521 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
    11581 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
    11641 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
    11701 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
    11761 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
    11821 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
    11881 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
    11941 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
    12001 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
    12061 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
    12121 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
    12181 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
    12241 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
    12301 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
    12361 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
    12421 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
    12481 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
    12541 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
    12601 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
    12661 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
    12721 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
    12781 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
    12841 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
    12901 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
    12961 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
    13021 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
    13081 cagcagagat ccagtttggt taattaaggt accgagggcc tatttcccat gattccttca
    13141 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
    13201 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
    13261 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
    13321 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgtcggcct gcggagacgg
    13381 gg
//
