LOCUS       sequence-261409-       12563 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  H1 promoter with two Tet operator (TetO) sites expresses 
            customizable Spyo-guide; short EF1a promoter (EFS) expresses 
            SaurCas9, 2A provides TetR, and 2A provides blasticidin resistance..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12563)
  AUTHORS   DeWeirdt PC, Sangree AK, Hanna RE, Sanson KR, Hegde M, Strand C, 
            Persky NS, Doench JG
  TITLE     Genetic screens in isogenic mammalian cell lines without single cell
            cloning.
  JOURNAL   Nat Commun. 2020 Feb 6;11(1):752. doi: 10.1038/s41467-020-14620-6.
  PUBMED    32029722
REFERENCE   2  (bases 1 to 12563)
  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: "Nat 
            Commun."; date: "2020-02-6"; volume: "11(1):752. doi"; pages: " 
            10.1038/s41467-020-14620-6"
COMMENT     Sequence Label: pRDA_103
FEATURES             Location/Qualifiers
     source          1..12563
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /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    2203..2320
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2496..2590
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     2507..2526
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    2544..2562
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2572..2590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2616..2691
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2753..2964
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2972..2981
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2984..3004
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3029..6184
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             6185..6232
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6242..6298
                     /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             6308..6925
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(6381..6400)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             6935..6988
                     /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             6989..7387
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7412..8000
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7465..7485)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7883..7894)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8078..8311
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8337..8354)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8343..8567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8613..9041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8700..8719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8910..8931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9050..9070)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9055..9384
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9235..9370
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9297..9316
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9432..9479
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9498..9872
                     /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    10002..10123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10039..10058)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10093..10112
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10172..10188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10172..10188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10185..10207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10196..10212
                     /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(10220..10226,10227..10244,10245..10250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10265..10286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10403..10420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10574..11162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10654..10673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11333..12124,12125..12193))
                     /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     11956..11975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12194..12298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(12373..12392)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2101 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2161 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2221 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2281 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2341 cagagatcca gtttggttaa ttaaggtacc cgaacgctga cgtcatcaac ccgctccaag
     2401 gaatcgcggg cccagtgtca ctaggcggga acacccagcg cgcgtgcgcc ctggcaggaa
     2461 gatggctgtg agggacaggg gagtggcgcc ctgcaatatt tgcatgtcgc tatgtgttct
     2521 gggaaatcac cataaacgtg aaatccctat cagtgataga gacttataag ttccctatca
     2581 gtgatagaga caccggagac gcttttttcg tctcagtttg agagctagaa atagcaagtt
     2641 caaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttgaa
     2701 ttcgctagct aggtcttgaa aggagtggga attggctccg gtgcccgtca gtgggcagag
     2761 cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg atccggtgcc
     2821 tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt
     2881 cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc
     2941 aacgggtttg ccgccagaac acaggaccgg tgccaccatg gcacccaaga agaagcgcaa
     3001 agttggcatc cacggcgtgc ctgctgccaa gcgcaactac atcctgggcc tggacattgg
     3061 catcacctca gttggctatg gcatcatcga ctacgagacc cgggacgtca tcgacgctgg
     3121 cgtccgcctc ttcaaggagg ccaacgtcga gaacaacgag ggccggcggt ccaagcgcgg
     3181 ggcccgccgg ctcaagcgcc gccgccgcca ccgcatccag cgcgtgaaaa agttgctctt
     3241 cgactacaac ttgctcaccg accactccga gctgtctggc atcaacccgt acgaggcccg
     3301 ggtcaagggc ctctcgcaaa agttgtccga ggaggagttc tctgctgccc tgctgcacct
     3361 ggccaagcgc cgcggggtcc acaacgtcaa cgaagttgag gaggacaccg gcaacgaact
     3421 ttccaccaag gagcagatct cccgcaactc caaggccctg gaggagaagt acgtcgcgga
     3481 gctccaactt gagcgcctca agaaggacgg cgaggtgcgc ggctccatca accgcttcaa
     3541 gacctccgac tacgtcaagg aggccaagca gctgctcaag gtccagaagg cctaccacca
     3601 acttgaccag tccttcatcg acacctacat cgacctgctg gagacccgcc gcacctacta
     3661 cgaggggcct ggcgagggct ctcccttcgg ctggaaggac atcaaggagt ggtacgagat
     3721 gctcatgggc cactgcacct acttcccgga ggagctgcgc tcggtcaagt acgcctacaa
     3781 cgccgacctc tacaacgcgc tcaacgacct caacaacttg gtcatcaccc gcgacgagaa
     3841 cgaaaagttg gagtactacg aaaagttcca gatcatcgaa aacgtcttca agcagaagaa
     3901 gaagccaact ttgaagcaga tcgccaagga gatcctggtc aacgaggagg acatcaaggg
     3961 ctaccgggtc acctccaccg gcaagcccga gttcaccaac ttaaaggtct accatgacat
     4021 caaggacatc accgcccgca aggagatcat cgagaacgcg gagctgctgg accagatcgc
     4081 caagatcctc accatctacc aaagttccga ggacatccag gaggagctga ccaacttaaa
     4141 ctcggagctt acccaggagg agatcgagca gatctccaac ttaaagggct acactggcac
     4201 ccacaacttg tccctcaagg ccatcaactt gatcctggac gaactttggc ataccaacga
     4261 caaccagatc gccatcttca accgactaaa gttggtgccc aagaaagttg acctgtccca
     4321 acagaaggag atcccaacta ccctggtcga cgacttcatc ctctcgcccg tggtcaagcg
     4381 cagcttcatc cagtccatca aggtcatcaa cgccatcatc aagaagtacg gcctgcccaa
     4441 cgacatcatc atcgagttgg cacgcgagaa gaactccaag gatgcccaga agatgatcaa
     4501 cgagatgcag aagcgcaacc gccagaccaa cgagcgcatc gaggagatca tccgcaccac
     4561 cggcaaggag aacgccaagt acctgatcga gaagataaag ttgcatgaca tgcaggaggg
     4621 caagtgcctc tactccctgg aggccatccc cctcgaggac ctgctcaaca accccttcaa
     4681 ctacgaagtt gaccacatca tcccgcgctc ggtgtccttc gacaacagct tcaacaacaa
     4741 ggtcctggtc aagcaggagg agaactccaa gaagggcaac cggaccccct tccagtacct
     4801 gtcctcctcg gactccaaga tctcctacga aactttcaag aagcacatcc tcaacttggc
     4861 aaagggcaag ggccgcatct ccaagaccaa gaaggagtac ctgctggagg agcgcgacat
     4921 caaccgcttc tcggtgcaga aggacttcat caaccgcaac ctggtggaca cccgctacgc
     4981 aacccgtggc ctcatgaacc tgctgcgcag ctacttccgg gtcaacaact tggacgtcaa
     5041 agttaagtcg atcaacggtg gcttcaccag cttcctgcgc cgcaagtgga agttcaagaa
     5101 ggagcgcaac aagggctaca agcaccatgc ggaggacgcc ctcatcatcg ccaacgccga
     5161 cttcatcttc aaggagtgga aaaagttgga caaggccaag aaggtcatgg agaaccagat
     5221 gttcgaggag aagcaggccg agtccatgcc ggagatcgag acagagcagg agtacaagga
     5281 gatcttcatc acgccgcacc agatcaagca catcaaggac ttcaaggact acaagtactc
     5341 ccaccgcgtg gacaagaagc ccaaccgcga gctcatcaac gacaccctct actccacccg
     5401 caaggacgac aagggcaaca ccctcatcgt caacaactta aacggcctct acgacaagga
     5461 caacgacaag ttgaaaaagt tgatcaacaa gagccccgaa aagttgctca tgtaccacca
     5521 tgacccgcag acgtaccaaa agttgaagtt gatcatggag cagtacggtg acgagaagaa
     5581 cccgctctac aagtactacg aggagaccgg caactacctc accaagtact ccaagaagga
     5641 caacggcccg gtcatcaaga agatcaagta ctacggcaac aagttgaatg cccacctgga
     5701 catcaccgac gactacccca actcccgcaa caaagttgta aagttgtcgc taaagccgta
     5761 ccgcttcgac gtctacctgg acaacggcgt ctacaagttc gtgaccgtca agaacctgga
     5821 cgtcatcaag aaggagaact actacgaggt caactccaag tgctacgagg aggccaaaaa
     5881 gttgaagaag atctccaacc aggccgagtt catcgccagc ttctacaaca acgacctcat
     5941 caagatcaac ggcgagctct accgcgtcat cggggtcaac aacgacctgc tgaaccgcat
     6001 cgaggtcaac atgatcgaca tcacctaccg cgagtacctc gagaacatga acgacaagcg
     6061 cccgcctcgc atcatcaaga ccatcgcctc caagacccag tccatcaaga agtactccac
     6121 cgacatcctt ggcaacttgt acgaggtcaa gagcaagaag caccctcaga tcatcaagaa
     6181 gggcaagcgc cctgctgcca ccaagaaggc tggccaggcc aagaagaaga agggatccgg
     6241 ggccaccaac ttttccctgc tgaagcaggc tggcgacgtc gaggagaacc ctggccctgg
     6301 ctccggaatg tccagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     6361 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     6421 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
     6481 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
     6541 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     6601 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     6661 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     6721 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     6781 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     6841 tcaccaaggc gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
     6901 aaaacaactt aaatgtgaaa gtgggggcag tggagagggc agaggaagtc tgctaacatg
     6961 cggtgacgtc gaggagaatc ctggcccaat ggccaagcct ttgtctcaag aagaatccac
     7021 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     7081 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     7141 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     7201 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     7261 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     7321 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     7381 gggctgataa tctagaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     7441 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     7501 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     7561 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     7621 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     7681 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     7741 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     7801 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     7861 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     7921 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     7981 cctttgggcc gcctccccgc agcgctgtcg actttaagac caatgactta caaggcagct
     8041 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     8101 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     8161 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     8221 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     8281 cccttttagt cagtgtggaa aatctctagc agggcccgtt taaacccgct gatcagcctc
     8341 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     8401 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     8461 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     8521 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     8581 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     8641 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     8701 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     8761 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     8821 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     8881 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     8941 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     9001 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     9061 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     9121 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     9181 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     9241 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     9301 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     9361 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     9421 gatcagcacg tgttgacaat taatcatcgg catagtatat cggcatagta taatacgaca
     9481 aggtgaggaa ctaaaccatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg
     9541 acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg
     9601 aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg
     9661 accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt
     9721 acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga
     9781 ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact
     9841 gcgtgcactt cgtggccgag gagcaggact gacacgtgct acgagatttc gattccaccg
     9901 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     9961 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt
    10021 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
    10081 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt
    10141 cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
    10201 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
    10261 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
    10321 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
    10381 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
    10441 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
    10501 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
    10561 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
    10621 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
    10681 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
    10741 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
    10801 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
    10861 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
    10921 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
    10981 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
    11041 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
    11101 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
    11161 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
    11221 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
    11281 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
    11341 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
    11401 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
    11461 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
    11521 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
    11581 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
    11641 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
    11701 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
    11761 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
    11821 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
    11881 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
    11941 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
    12001 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
    12061 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
    12121 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
    12181 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
    12241 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
    12301 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
    12361 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
    12421 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
    12481 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
    12541 tgtacgggcc agatatacgc gtt
//
