LOCUS       sequence-310691-       13374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCas9-P2A-EGFP driven by human SYN promoter and empty 
            CRISPR Display sgRNA/accessory RNA from U6 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13374)
  AUTHORS   Carullo NVN, Phillips Iii RA, Simon RC, Soto SAR, Hinds JE, 
            Salisbury AJ, Revanna JS, Bunner KD, Ianov L, Sultan FA, Savell KE, 
            Gersbach CA, Day JJ
  TITLE     Enhancer RNAs predict enhancer-gene regulatory links and are 
            critical for enhancer function in neuronal systems.
  JOURNAL   Nucleic Acids Res. 2020 Aug 18. pii: 5893972. doi: 
            10.1093/nar/gkaa671.
  PUBMED    32810208
REFERENCE   2  (bases 1 to 13374)
  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: "Nucleic 
            Acids Res. 2020 Aug 18. pii: 5893972. doi: 10.1093/nar/gkaa671."
COMMENT     Sequence Label: lenti U6-sgRNA-acRNA SYN-dCas9-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..13374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        230..609
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     764..784
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             827..1007
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1054..1179
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1672..1905
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2090..2134
                     /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    2432..2549
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2602..2622
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2604..2622
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_RNA        complement(2664..2739)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2758..3006)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2816..2835)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2986..3006)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        3084..3531
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      3549..3558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3555..3581,3582..3584,3585..6071,6072..6074,
                     6075..7658)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3555..7658
                     /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
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7659..7706
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7740..7796
                     /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             join(7800..7802,7803..7805,7806..8519)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(7800..7802,7803..7805,7806..8516)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8106..8125)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8453..8474
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    8544..9132
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8597..8617)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9015..9026)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9135..9151)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9136..9152)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9171..9351
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9377..9394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9383..9607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9653..10081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9740..9759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9950..9971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10090..10110)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10095..10424
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10275..10410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10337..10356
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10472..10519
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10538..10912
                     /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    11042..11163
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11079..11098)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11133..11152
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11212..11228)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11212..11228)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11225..11247)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11236..11252
                     /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(11260..11266,11267..11284,11285..11290))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11305..11326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11443..11460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11614..12202)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11694..11713)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12373..13164,13165..13233))
                     /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     12996..13015
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13234..13338)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
       61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      781 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
      841 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      901 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
      961 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     1021 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     1081 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     1141 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     1201 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     1261 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     1321 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1381 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1441 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1501 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa
     1561 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1621 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     1681 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     1741 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     1801 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     1861 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     1921 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc
     1981 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     2041 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     2101 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     2161 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     2221 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     2281 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     2341 agagagagac agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat
     2401 tctgcagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     2461 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     2521 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag
     2581 tttggttaat taaccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc
     2641 caccgcggaa aaaaacagag acggcaccga ctcggtgcca ctttttcaag ttgataacgg
     2701 actagcctta ttttaacttg ctatttctag ctctaaaaca ggtcttctcg aagacccggt
     2761 gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
     2821 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
     2881 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc
     2941 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
     3001 gccctcggta ccttaacccg tgtcggctgc tagctaggtc ttgaaaggag tgggaattgg
     3061 ctccggtgcc cgtcagtttc gaaagtgcaa gtgggtttta ggaccaggat gaggcggggt
     3121 gggggtgcct acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc
     3181 caaattgcgc atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc
     3241 gcactgccag cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac
     3301 cgccgcctca gcactgaagg cgcgctgacg tcactcgccg gtcccccgca aactcccctt
     3361 cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg
     3421 cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg
     3481 cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca gaccggttct
     3541 agagcgctgc caccatggac aagaagtaca gcatcggcct ggccatcggc accaactctg
     3601 tgggctgggc cgtgatcacc gacgagtaca aggtgcccag caagaaattc aaggtgctgg
     3661 gcaacaccga ccggcacagc atcaagaaga acctgatcgg agccctgctg ttcgacagcg
     3721 gcgaaacagc cgaggccacc cggctgaaga gaaccgccag aagaagatac accagacgga
     3781 agaaccggat ctgctatctg caagagatct tcagcaacga gatggccaag gtggacgaca
     3841 gcttcttcca cagactggaa gagtccttcc tggtggaaga ggataagaag cacgagcggc
     3901 accccatctt cggcaacatc gtggacgagg tggcctacca cgagaagtac cccaccatct
     3961 accacctgag aaagaaactg gtggacagca ccgacaaggc cgacctgcgg ctgatctatc
     4021 tggccctggc ccacatgatc aagttccggg gccacttcct gatcgagggc gacctgaacc
     4081 ccgacaacag cgacgtggac aagctgttca tccagctggt gcagacctac aaccagctgt
     4141 tcgaggaaaa ccccatcaac gccagcggcg tggacgccaa ggccatcctg tctgccagac
     4201 tgagcaagag cagacggctg gaaaatctga tcgcccagct gcccggcgag aagaagaatg
     4261 gcctgttcgg caacctgatt gccctgagcc tgggcctgac ccccaacttc aagagcaact
     4321 tcgacctggc cgaggatgcc aaactgcagc tgagcaagga cacctacgac gacgacctgg
     4381 acaacctgct ggcccagatc ggcgaccagt acgccgacct gtttctggcc gccaagaacc
     4441 tgtccgacgc catcctgctg agcgacatcc tgagagtgaa caccgagatc accaaggccc
     4501 ccctgagcgc ctctatgatc aagagatacg acgagcacca ccaggacctg accctgctga
     4561 aagctctcgt gcggcagcag ctgcctgaga agtacaaaga gattttcttc gaccagagca
     4621 agaacggcta cgccggctac attgacggcg gagccagcca ggaagagttc tacaagttca
     4681 tcaagcccat cctggaaaag atggacggca ccgaggaact gctcgtgaag ctgaacagag
     4741 aggacctgct gcggaagcag cggaccttcg acaacggcag catcccccac cagatccacc
     4801 tgggagagct gcacgccatt ctgcggcggc aggaagattt ttacccattc ctgaaggaca
     4861 accgggaaaa gatcgagaag atcctgacct tccgcatccc ctactacgtg ggccctctgg
     4921 ccaggggaaa cagcagattc gcctggatga ccagaaagag cgaggaaacc atcaccccct
     4981 ggaacttcga ggaagtggtg gacaagggcg cttccgccca gagcttcatc gagcggatga
     5041 ccaacttcga taagaacctg cccaacgaga aggtgctgcc caagcacagc ctgctgtacg
     5101 agtacttcac cgtgtataac gagctgacca aagtgaaata cgtgaccgag ggaatgagaa
     5161 agcccgcctt cctgagcggc gagcagaaaa aggccatcgt ggacctgctg ttcaagacca
     5221 accggaaagt gaccgtgaag cagctgaaag aggactactt caagaaaatc gagtgcttcg
     5281 actccgtgga aatctccggc gtggaagatc ggttcaacgc ctccctgggc acataccacg
     5341 atctgctgaa aattatcaag gacaaggact tcctggacaa tgaggaaaac gaggacattc
     5401 tggaagatat cgtgctgacc ctgacactgt ttgaggacag agagatgatc gaggaacggc
     5461 tgaaaaccta tgcccacctg ttcgacgaca aagtgatgaa gcagctgaag cggcggagat
     5521 acaccggctg gggcaggctg agccggaagc tgatcaacgg catccgggac aagcagtccg
     5581 gcaagacaat cctggatttc ctgaagtccg acggcttcgc caacagaaac ttcatgcagc
     5641 tgatccacga cgacagcctg acctttaaag aggacatcca gaaagcccag gtgtccggcc
     5701 agggcgatag cctgcacgag cacattgcca atctggccgg cagccccgcc attaagaagg
     5761 gcatcctgca gacagtgaag gtggtggacg agctcgtgaa agtgatgggc cggcacaagc
     5821 ccgagaacat cgtgatcgaa atggccagag agaaccagac cacccagaag ggacagaaga
     5881 acagccgcga gagaatgaag cggatcgaag agggcatcaa agagctgggc agccagatcc
     5941 tgaaagaaca ccccgtggaa aacacccagc tgcagaacga gaagctgtac ctgtactacc
     6001 tgcagaatgg gcgggatatg tacgtggacc aggaactgga catcaaccgg ctgtccgact
     6061 acgatgtgga cgctatcgtg cctcagagct ttctgaagga cgactccatc gacaacaagg
     6121 tgctgaccag aagcgacaag aaccggggca agagcgacaa cgtgccctcc gaagaggtcg
     6181 tgaagaagat gaagaactac tggcggcagc tgctgaacgc caagctgatt acccagagaa
     6241 agttcgacaa tctgaccaag gccgagagag gcggcctgag cgaactggat aaggccggct
     6301 tcatcaagag acagctggtg gaaacccggc agatcacaaa gcacgtggca cagatcctgg
     6361 actcccggat gaacactaag tacgacgaga atgacaagct gatccgggaa gtgaaagtga
     6421 tcaccctgaa gtccaagctg gtgtccgatt tccggaagga tttccagttt tacaaagtgc
     6481 gcgagatcaa caactaccac cacgcccacg acgcctacct gaacgccgtc gtgggaaccg
     6541 ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt gtacggcgac tacaaggtgt
     6601 acgacgtgcg gaagatgatc gccaagagcg agcaggaaat cggcaaggct accgccaagt
     6661 acttcttcta cagcaacatc atgaactttt tcaagaccga gattaccctg gccaacggcg
     6721 agatccggaa gcggcctctg atcgagacaa acggcgaaac cggggagatc gtgtgggata
     6781 agggccggga ttttgccacc gtgcggaaag tgctgagcat gccccaagtg aatatcgtga
     6841 aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc tatcctgccc aagaggaaca
     6901 gcgataagct gatcgccaga aagaaggact gggaccctaa gaagtacggc ggcttcgaca
     6961 gccccaccgt ggcctattct gtgctggtgg tggccaaagt ggaaaagggc aagtccaaga
     7021 aactgaagag tgtgaaagag ctgctgggga tcaccatcat ggaaagaagc agcttcgaga
     7081 agaatcccat cgactttctg gaagccaagg gctacaaaga agtgaaaaag gacctgatca
     7141 tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg ccggaagaga atgctggcct
     7201 ctgccggcga actgcagaag ggaaacgaac tggccctgcc ctccaaatat gtgaacttcc
     7261 tgtacctggc cagccactat gagaagctga agggctcccc cgaggataat gagcagaaac
     7321 agctgtttgt ggaacagcac aagcactacc tggacgagat catcgagcag atcagcgagt
     7381 tctccaagag agtgatcctg gccgacgcta atctggacaa agtgctgtcc gcctacaaca
     7441 agcaccggga taagcccatc agagagcagg ccgagaatat catccacctg tttaccctga
     7501 ccaatctggg agcccctgcc gccttcaagt actttgacac caccatcgac cggaagaggt
     7561 acaccagcac caaagaggtg ctggacgcca ccctgatcca ccagagcatc accggcctgt
     7621 acgagacacg gatcgacctg tctcagctgg gaggcgacaa gcgacctgcc gccacaaaga
     7681 aggctggaca ggctaagaag aagaaagatt acaaagacga tggcgataag ggatccggcg
     7741 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgacca
     7801 tggtgagcaa aggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     7861 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     7921 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     7981 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     8041 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     8101 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     8161 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     8221 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     8281 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     8341 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     8401 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     8461 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag
     8521 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     8581 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     8641 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     8701 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     8761 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     8821 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     8881 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     8941 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     9001 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     9061 ctctgcggcc tcttccgcct cttcgccttc gccctcagac gagtcggatc tccctttggg
     9121 ccgcctcccc gcatcgatac cgtcgacctc gagaccttcc ggactgtact gggtctctct
     9181 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     9241 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     9301 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agggcccgtt
     9361 taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     9421 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     9481 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     9541 caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga tgcggtgggc
     9601 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     9661 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     9721 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     9781 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     9841 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     9901 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     9961 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
    10021 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
    10081 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
    10141 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
    10201 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
    10261 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
    10321 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
    10381 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
    10441 gtatatccat tttcggatct gatcagcacg tgttgacaat taatcatcgg catagtatat
    10501 cggcatagta taatacgaca aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt
    10561 tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg
    10621 gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct
    10681 gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt
    10741 gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga
    10801 cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct
    10861 gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gacacgtgct
    10921 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
    10981 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
    11041 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
    11101 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
    11161 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
    11221 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
    11281 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
    11341 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
    11401 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
    11461 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
    11521 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
    11581 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
    11641 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
    11701 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
    11761 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
    11821 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
    11881 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
    11941 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
    12001 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
    12061 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
    12121 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
    12181 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
    12241 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
    12301 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
    12361 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
    12421 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
    12481 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
    12541 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
    12601 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
    12661 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
    12721 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
    12781 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
    12841 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
    12901 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
    12961 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
    13021 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
    13081 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
    13141 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
    13201 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
    13261 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
    13321 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acgg
//
