LOCUS       sequence-486087-       15582 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Mammalian expression of dEnAsCas12a-VPR with EGFP selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 15582)
  AUTHORS   Melore SM, Hamilton MC, Reddy TE
  TITLE     HyperCas12a enables highly-multiplexed epigenome editing screens.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 9:2024.07.08.602263. doi: 
            10.1101/2024.07.08.602263.
  PUBMED    39026853
REFERENCE   2  (bases 1 to 15582)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Jul 9:2024.07.08.602263. doi: 
            10.1101/2024.07.08.602263."
COMMENT     Sequence Label: SM283_EF1A-dEnAsCas12a-VPR-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..15582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1680..1913
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2098..2142
                     /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    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2630..3808
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2861..3799
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3756..3776
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             3824..7744
                     /codon_start=1
                     /product="CRISPR-associated protein from Acidaminococcus 
                     sp. BV3L6 (Zetsche et al., 2015)"
                     /label=AsCpf1
                     /translation="MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND
                     HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRN
                     AIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD
                     KFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHF
                     ENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVL
                     NLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLL
                     RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTG
                     KITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL
                     KKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKA
                     RNYATKKPYSVEKFKLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYK
                     ALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEP
                     LEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSID
                     LSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKG
                     HHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLK
                     DQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFF
                     HVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIARGERNLIYITVIDSTGKILEQR
                     SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV
                     VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQ
                     FTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD
                     VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIEN
                     HRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM
                     RNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKES
                     KDLKLQNGISNQDWLAYIQELRN"
     CDS             7751..7777
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7757..7783
                     /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             7796..7822
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             7829..7855
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             7892..8041
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             8066..8086
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8516..8872
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             8891..9460
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             9467..9520
                     /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             join(9527..9529,9530..10240)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(9527..9529,9530..10240)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(9569..9590)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     10177..10198
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    10274..10862
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10327..10347)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10745..10756)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(10865..10881)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10866..10882)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             11387..11567
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(11593..11610)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    11599..11823
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      11869..12297
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11956..11975)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     12166..12187
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(12306..12326)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        12311..12640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      12491..12626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     12553..12572
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        12688..12735
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             12754..13128
                     /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    13258..13379
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13295..13314)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13349..13368
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(13428..13444)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13428..13444)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(13441..13463)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    13452..13468
                     /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(13476..13482,13483..13500,13501..13506))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    13521..13542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(13659..13676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(13830..14418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(13910..13929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(14589..15380,15381..15449))
                     /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     15212..15231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(15450..15554)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
     1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
     2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     2581 agatccagtt tggttaatta acccgtgtcg gctccagatc tggcctccgg gctccggtgc
     2641 ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     2701 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     2761 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     2821 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc
     2881 ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta cttccacctg
     2941 gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag
     3001 gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg
     3061 gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc
     3121 tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct
     3181 tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga
     3241 cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc
     3301 gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc
     3361 gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc
     3421 ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc
     3481 gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt
     3541 cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctcgag
     3601 cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca
     3661 cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga
     3721 atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag
     3781 tttttttctt ccatttcagg tgtcgtgagc ttctagagcc accatgacac agttcgaggg
     3841 ctttaccaac ctgtatcagg tgagcaagac actgcggttt gagctgatcc cacagggcaa
     3901 gaccctgaag cacatccagg agcagggctt catcgaggag gacaaggccc gcaatgatca
     3961 ctacaaggag ctgaagccca tcatcgatcg gatctacaag acctatgccg accagtgcct
     4021 gcagctggtg cagctggatt gggagaacct gagcgccgcc atcgactcct atagaaagga
     4081 gaaaaccgag gagacaagga acgccctgat cgaggagcag gccacatatc gcaatgccat
     4141 ccacgactac ttcatcggcc ggacagacaa cctgaccgat gccatcaata agagacacgc
     4201 cgagatctac aagggcctgt tcaaggccga gctgtttaat ggcaaggtgc tgaagcagct
     4261 gggcaccgtg accacaaccg agcacgagaa cgccctgctg cggagcttcg acaagtttac
     4321 aacctacttc tccggctttt atagaaacag gaagaacgtg ttcagcgccg aggatatcag
     4381 cacagccatc ccacaccgca tcgtgcagga caacttcccc aagtttaagg agaattgtca
     4441 catcttcaca cgcctgatca ccgccgtgcc cagcctgcgg gagcactttg agaacgtgaa
     4501 gaaggccatc ggcatcttcg tgagcacctc catcgaggag gtgttttcct tcccttttta
     4561 taaccagctg ctgacacaga cccagatcga cctgtataac cagctgctgg gaggaatctc
     4621 tcgggaggca ggcaccgaga agatcaaggg cctgaacgag gtgctgaatc tggccatcca
     4681 gaagaatgat gagacagccc acatcatcgc ctccctgcca cacagattca tccccctgtt
     4741 taagcagatc ctgtccgata ggaacaccct gtctttcatc ctggaggagt ttaagagcga
     4801 cgaggaagtg atccagtcct tctgcaagta caagacactg ctgagaaacg agaacgtgct
     4861 ggagacagcc gaggccctgt ttaacgagct gaacagcatc gacctgacac acatcttcat
     4921 cagccacaag aagctggaga caatcagcag cgccctgtgc gaccactggg atacactgag
     4981 gaatgccctg tatgagcgga gaatctccga gctgacaggc aagatcacca agtctgccaa
     5041 ggagaaggtg cagcgcagcc tgaagcacga ggatatcaac ctgcaggaga tcatctctgc
     5101 cgcaggcaag gagctgagcg aggccttcaa gcagaaaacc agcgagatcc tgtcccacgc
     5161 acacgccgcc ctggatcagc cactgcctac aaccctgaag aagcaggagg agaaggagat
     5221 cctgaagtct cagctggaca gcctgctggg cctgtaccac ctgctggact ggtttgccgt
     5281 ggatgagtcc aacgaggtgg accccgagtt ctctgcccgg ctgaccggca tcaagctgga
     5341 gatggagcct tctctgagct tctacaacaa ggccagaaat tatgccacca agaagcccta
     5401 ctccgtggag aagttcaagc tgaactttca gatgcctaca ctggccagag gctgggacgt
     5461 gaatagagag aagaacaatg gcgccatcct gtttgtgaag aacggcctgt actatctggg
     5521 catcatgcca aagcagaagg gcaggtataa ggccctgagc ttcgagccca cagagaaaac
     5581 cagcgagggc tttgataaga tgtactatga ctacttccct gatgccgcca agatgatccc
     5641 aaagtgcagc acccagctga aggccgtgac agcccacttt cagacccaca caacccccat
     5701 cctgctgtcc aacaatttca tcgagcctct ggagatcaca aaggagatct acgacctgaa
     5761 caatcctgag aaggagccaa agaagtttca gacagcctac gccaagaaaa ccggcgacca
     5821 gaagggctac agagaggccc tgtgcaagtg gatcgacttc acaagggatt ttctgtccaa
     5881 gtataccaag acaacctcta tcgatctgtc tagcctgcgg ccatcctctc agtataagga
     5941 cctgggcgag tactatgccg agctgaatcc cctgctgtac cacatcagct tccagagaat
     6001 cgccgagaag gagatcatgg atgccgtgga gacaggcaag ctgtacctgt tccagatcta
     6061 taacaaggac tttgccaagg gccaccacgg caagcctaat ctgcacacac tgtattggac
     6121 cggcctgttt tctccagaga acctggccaa gacaagcatc aagctgaatg gccaggccga
     6181 gctgttctac cgccctaagt ccaggatgaa gaggatggca caccggctgg gagagaagat
     6241 gctgaacaag aagctgaagg atcagaaaac cccaatcccc gacaccctgt accaggagct
     6301 gtacgactat gtgaatcaca gactgtccca cgacctgtct gatgaggcca gggccctgct
     6361 gcccaacgtg atcaccaagg aggtgtctca cgagatcatc aaggataggc gctttaccag
     6421 cgacaagttc tttttccacg tgcctatcac actgaactat caggccgcca attccccatc
     6481 taagttcaac cagagggtga atgcctacct gaaggagcac cccgagacac ctatcatcgg
     6541 catcgcccgg ggcgagagaa acctgatcta tatcacagtg atcgactcca ccggcaagat
     6601 cctggagcag cggagcctga acaccatcca gcagtttgat taccagaaga agctggacaa
     6661 cagggagaag gagagggtgg cagcaaggca ggcctggtct gtggtgggca caatcaagga
     6721 tctgaagcag ggctatctga gccaggtcat ccacgagatc gtggacctga tgatccacta
     6781 ccaggccgtg gtggtgctgg agaacctgaa tttcggcttt aagagcaaga ggaccggcat
     6841 cgccgagaag gccgtgtacc agcagttcga gaagatgctg atcgataagc tgaattgcct
     6901 ggtgctgaag gactatccag cagagaaagt gggaggcgtg ctgaacccat accagctgac
     6961 agaccagttc acctcctttg ccaagatggg cacccagtct ggcttcctgt tttacgtgcc
     7021 tgccccatat acatctaaga tcgatcccct gaccggcttc gtggacccct tcgtgtggaa
     7081 aaccatcaag aatcacgaga gccgcaagca cttcctggag ggcttcgact ttctgcacta
     7141 cgacgtgaaa accggcgact tcatcctgca ctttaagatg aacagaaatc tgtccttcca
     7201 gaggggcctg cccggcttta tgcctgcatg ggatatcgtg ttcgagaaga acgagacaca
     7261 gtttgacgcc aagggcaccc ctttcatcgc cggcaagaga atcgtgccag tgatcgagaa
     7321 tcacagattc accggcagat accgggacct gtatcctgcc aacgagctga tcgccctgct
     7381 ggaggagaag ggcatcgtgt tcagggatgg ctccaacatc ctgccaaagc tgctggagaa
     7441 tgacgattct cacgccatcg acacgatggt ggccctgatc cgcagcgtgc tgcagatgcg
     7501 gaactccaat gccgccacag gcgaggacta tatcaacagc cccgtgcgcg atctgaatgg
     7561 cgtgtgcttc gactcccggt ttcagaaccc agagtggcca atggacgccg atgccaatgg
     7621 cgcctaccac atcgccctga agggccagct gctgctgaat cacctgaagg agagcaagga
     7681 tctgaagctg cagaacggca tctccaatca ggactggctg gcctacatcc aggagctgcg
     7741 caacggatcc tatccctatg acgtgcccga ttatgccagc ctgggcagcg gctcccctgc
     7801 tgccaaacgc gttaaactag acgaagatcc tgccgcgaag cgcgtgaaac tggacgacgg
     7861 cattggtagt gggagcaacg gcagcagcct cgacgcattg gacgattttg atctggatat
     7921 gctgggaagt gacgccctcg atgattttga ccttgacatg cttggttcgg atgcccttga
     7981 tgactttgac ctcgacatgc tcggcagtga cgcccttgat gatttcgacc tggacatgct
     8041 gattaactct agaagttccg gatctccgaa aaagaaacgc aaagttggta gccagtacct
     8101 gcccgacacc gacgaccggc accggatcga ggaaaagcgg aagcggacct acgagacatt
     8161 caagagcatc atgaagaagt cccccttcag cggccccacc gaccctagac ctccacctag
     8221 aagaatcgcc gtgcccagca gatccagcgc cagcgtgcca aaacctgccc cccagcctta
     8281 ccccttcacc agcagcctga gcaccatcaa ctacgacgag ttccctacca tggtgttccc
     8341 cagcggccag atctctcagg cctctgctct ggctccagcc cctcctcagg tgctgcctca
     8401 ggctcctgct cctgcaccag ctccagccat ggtgtctgca ctggctcagg caccagcacc
     8461 cgtgcctgtg ctggctcctg gacctccaca ggctgtggct ccaccagccc ctaaacctac
     8521 acaggccggc gagggcacac tgtctgaagc tctgctgcag ctgcagttcg acgacgagga
     8581 tctgggagcc ctgctgggaa acagcaccga tcctgccgtg ttcaccgacc tggccagcgt
     8641 ggacaacagc gagttccagc agctgctgaa ccagggcatc cctgtggccc ctcacaccac
     8701 cgagcccatg ctgatggaat accccgaggc catcacccgg ctcgtgacag gcgctcagag
     8761 gcctcctgat ccagctcctg cccctctggg agcaccaggc ctgcctaatg gactgctgtc
     8821 tggcgacgag gacttcagct ctatcgccga tatggatttc tcagccttgc tgggctctgg
     8881 cagcggcagc cgggattcca gggaagggat gtttttgccg aagcctgagg ccggctccgc
     8941 tattagtgac gtgtttgagg gccgcgaggt gtgccagcca aaacgaatcc ggccatttca
     9001 tcctccagga agtccatggg ccaaccgccc actccccgcc agcctcgcac caacaccaac
     9061 cggtccagta catgagccag tcgggtcact gaccccggca ccagtccctc agccactgga
     9121 tccagcgccc gcagtgactc ccgaggccag tcacctgttg gaggatcccg atgaagagac
     9181 gagccaggct gtcaaagccc ttcgggagat ggccgatact gtgattcccc agaaggaaga
     9241 ggctgcaatc tgtggccaaa tggacctttc ccatccgccc ccaaggggcc atctggatga
     9301 gctgacaacc acacttgagt ccatgaccga ggatctgaac ctggactcac ccctgacccc
     9361 ggaattgaac gagattctgg ataccttcct gaacgacgag tgcctcttgc atgccatgca
     9421 tatcagcaca ggactgtcca tcttcgacac atctctgttt gctagcgagg gcagaggaag
     9481 tcttctaaca tgcggtgacg tggaggagaa tcccggccct ggtaccgtga gcaagggcga
     9541 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     9601 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     9661 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     9721 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     9781 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     9841 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     9901 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     9961 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
    10021 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
    10081 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
    10141 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
    10201 cgccgccggg atcactctcg gcatggacga gctgtacaag accggttgag aattcgatat
    10261 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
    10321 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
    10381 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
    10441 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
    10501 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
    10561 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
    10621 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
    10681 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
    10741 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
    10801 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
    10861 gcatcgatac cgtcgacctc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
    10921 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
    10981 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
    11041 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
    11101 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
    11161 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
    11221 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
    11281 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
    11341 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
    11401 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
    11461 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
    11521 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa
    11581 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
    11641 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
    11701 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
    11761 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
    11821 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
    11881 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
    11941 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
    12001 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
    12061 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
    12121 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
    12181 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
    12241 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat
    12301 tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag
    12361 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
    12421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
    12481 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
    12541 actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa
    12601 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
    12661 atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata gtatatcggc
    12721 atagtataat acgacaaggt gaggaactaa accatggcca agttgaccag tgccgttccg
    12781 gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc
    12841 tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc
    12901 atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc
    12961 ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc
    13021 tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc
    13081 gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctacga
    13141 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
    13201 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
    13261 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
    13321 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
    13381 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
    13441 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
    13501 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
    13561 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
    13621 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
    13681 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
    13741 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
    13801 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
    13861 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
    13921 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
    13981 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
    14041 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
    14101 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
    14161 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
    14221 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
    14281 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
    14341 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
    14401 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
    14461 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
    14521 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
    14581 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
    14641 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
    14701 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
    14761 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
    14821 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
    14881 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
    14941 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
    15001 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
    15061 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
    15121 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
    15181 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
    15241 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
    15301 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
    15361 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
    15421 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
    15481 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
    15541 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg ac
//
