LOCUS       sequence-486086-       13977 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Mammalian expression of dEnAsCas12a with EGFP selectable marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13977)
  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 13977)
  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: SM470_EF1A-dEnAsCas12a-T2A-EGFP
FEATURES             Location/Qualifiers
     source          1..13977
                     /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             7862..7915
                     /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(7922..7924,7925..8635)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(7922..7924,7925..8635)
                     /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(7964..7985)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8225..8244)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     8572..8593
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    8669..9257
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8722..8742)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(9140..9151)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(9260..9276)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9261..9277)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             9782..9962
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(9988..10005)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9994..10218
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      10264..10692
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10351..10370)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10561..10582
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10701..10721)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        10706..11035
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      10886..11021
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     10948..10967
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        11083..11130
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             11149..11523
                     /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    11653..11774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11690..11709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11744..11763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(11823..11839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(11823..11839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(11836..11858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    11847..11863
                     /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(11871..11877,11878..11895,11896..11901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    11916..11937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(12054..12071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(12225..12813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(12305..12324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12984..13775,13776..13844))
                     /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     13607..13626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(13845..13949)
                     /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 tggacgctag
     7861 cgagggcaga ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctggtac
     7921 cgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     7981 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     8041 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     8101 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     8161 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     8221 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     8281 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     8341 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     8401 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     8461 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     8521 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     8581 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagaccgg
     8641 ttgagaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     8701 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     8761 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     8821 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     8881 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     8941 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     9001 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     9061 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     9121 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     9181 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     9241 ttgggccgcc tccccgcatc gataccgtcg acctcgagac ctagaaaaac atggagcaat
     9301 cacaagtagc aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga
     9361 ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc
     9421 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     9481 ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga
     9541 ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta
     9601 caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg
     9661 cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg
     9721 gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc cggactgtac
     9781 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     9841 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     9901 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     9961 cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
    10021 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
    10081 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
    10141 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
    10201 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
    10261 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
    10321 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
    10381 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
    10441 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
    10501 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
    10561 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
    10621 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
    10681 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
    10741 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
    10801 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
    10861 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
    10921 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
    10981 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
    11041 cccgggagct tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg
    11101 gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg
    11161 accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc
    11221 gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac
    11281 gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc
    11341 tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg
    11401 aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg
    11461 gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac
    11521 tgacacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga
    11581 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc
    11641 ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
    11701 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
    11761 atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca
    11821 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
    11881 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
    11941 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
    12001 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
    12061 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
    12121 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
    12181 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
    12241 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
    12301 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
    12361 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
    12421 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
    12481 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
    12541 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
    12601 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
    12661 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
    12721 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
    12781 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
    12841 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
    12901 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
    12961 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
    13021 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
    13081 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
    13141 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
    13201 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
    13261 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
    13321 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
    13381 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
    13441 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
    13501 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
    13561 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
    13621 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
    13681 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
    13741 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
    13801 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
    13861 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
    13921 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgac
//
