LOCUS       sequence-256515-       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"
     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        2733..2827
                     /label=H1-2O2 promoter
                     /note="doxycycline-inducible variant of the human H1 RNA 
                     promoter (Henriksen et al., 2007)"
     primer_bind     2744..2763
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     protein_bind    2781..2799
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2809..2827
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        2853..2928
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2990..3201
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3209..3218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3221..3241
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3266..6421
                     /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             6422..6469
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6479..6535
                     /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             6545..7162
                     /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(6618..6637)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             7172..7225
                     /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             7226..7624
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    7649..8237
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(7702..7722)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8120..8131)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8315..8548
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(8574..8591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8580..8804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      8850..9278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8937..8956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9147..9168
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9287..9307)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9292..9621
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9472..9607
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9534..9553
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        9669..9716
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             9735..10109
                     /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    10239..10360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10276..10295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10330..10349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10409..10425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10409..10425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10422..10444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10433..10449
                     /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(10457..10463,10464..10481,10482..10487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10502..10523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10640..10657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10811..11399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10891..10910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(11570..12361,12362..12430))
                     /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     12193..12212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12431..12535)
                     /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 aggtacccga acgctgacgt catcaacccg ctccaaggaa
     2641 tcgcgggccc agtgtcacta ggcgggaaca cccagcgcgc gtgcgccctg gcaggaagat
     2701 ggctgtgagg gacaggggag tggcgccctg caatatttgc atgtcgctat gtgttctggg
     2761 aaatcaccat aaacgtgaaa tccctatcag tgatagagac ttataagttc cctatcagtg
     2821 atagagacac cggagacgct tttttcgtct cagtttgaga gctagaaata gcaagttcaa
     2881 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttgaattc
     2941 gctagctagg tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc
     3001 acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag
     3061 agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc
     3121 gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac
     3181 gggtttgccg ccagaacaca ggaccggtgc caccatggca cccaagaaga agcgcaaagt
     3241 tggcatccac ggcgtgcctg ctgccaagcg caactacatc ctgggcctgg acattggcat
     3301 cacctcagtt ggctatggca tcatcgacta cgagacccgg gacgtcatcg acgctggcgt
     3361 ccgcctcttc aaggaggcca acgtcgagaa caacgagggc cggcggtcca agcgcggggc
     3421 ccgccggctc aagcgccgcc gccgccaccg catccagcgc gtgaaaaagt tgctcttcga
     3481 ctacaacttg ctcaccgacc actccgagct gtctggcatc aacccgtacg aggcccgggt
     3541 caagggcctc tcgcaaaagt tgtccgagga ggagttctct gctgccctgc tgcacctggc
     3601 caagcgccgc ggggtccaca acgtcaacga agttgaggag gacaccggca acgaactttc
     3661 caccaaggag cagatctccc gcaactccaa ggccctggag gagaagtacg tcgcggagct
     3721 ccaacttgag cgcctcaaga aggacggcga ggtgcgcggc tccatcaacc gcttcaagac
     3781 ctccgactac gtcaaggagg ccaagcagct gctcaaggtc cagaaggcct accaccaact
     3841 tgaccagtcc ttcatcgaca cctacatcga cctgctggag acccgccgca cctactacga
     3901 ggggcctggc gagggctctc ccttcggctg gaaggacatc aaggagtggt acgagatgct
     3961 catgggccac tgcacctact tcccggagga gctgcgctcg gtcaagtacg cctacaacgc
     4021 cgacctctac aacgcgctca acgacctcaa caacttggtc atcacccgcg acgagaacga
     4081 aaagttggag tactacgaaa agttccagat catcgaaaac gtcttcaagc agaagaagaa
     4141 gccaactttg aagcagatcg ccaaggagat cctggtcaac gaggaggaca tcaagggcta
     4201 ccgggtcacc tccaccggca agcccgagtt caccaactta aaggtctacc atgacatcaa
     4261 ggacatcacc gcccgcaagg agatcatcga gaacgcggag ctgctggacc agatcgccaa
     4321 gatcctcacc atctaccaaa gttccgagga catccaggag gagctgacca acttaaactc
     4381 ggagcttacc caggaggaga tcgagcagat ctccaactta aagggctaca ctggcaccca
     4441 caacttgtcc ctcaaggcca tcaacttgat cctggacgaa ctttggcata ccaacgacaa
     4501 ccagatcgcc atcttcaacc gactaaagtt ggtgcccaag aaagttgacc tgtcccaaca
     4561 gaaggagatc ccaactaccc tggtcgacga cttcatcctc tcgcccgtgg tcaagcgcag
     4621 cttcatccag tccatcaagg tcatcaacgc catcatcaag aagtacggcc tgcccaacga
     4681 catcatcatc gagttggcac gcgagaagaa ctccaaggat gcccagaaga tgatcaacga
     4741 gatgcagaag cgcaaccgcc agaccaacga gcgcatcgag gagatcatcc gcaccaccgg
     4801 caaggagaac gccaagtacc tgatcgagaa gataaagttg catgacatgc aggagggcaa
     4861 gtgcctctac tccctggagg ccatccccct cgaggacctg ctcaacaacc ccttcaacta
     4921 cgaagttgac cacatcatcc cgcgctcggt gtccttcgac aacagcttca acaacaaggt
     4981 cctggtcaag caggaggaga actccaagaa gggcaaccgg acccccttcc agtacctgtc
     5041 ctcctcggac tccaagatct cctacgaaac tttcaagaag cacatcctca acttggcaaa
     5101 gggcaagggc cgcatctcca agaccaagaa ggagtacctg ctggaggagc gcgacatcaa
     5161 ccgcttctcg gtgcagaagg acttcatcaa ccgcaacctg gtggacaccc gctacgcaac
     5221 ccgtggcctc atgaacctgc tgcgcagcta cttccgggtc aacaacttgg acgtcaaagt
     5281 taagtcgatc aacggtggct tcaccagctt cctgcgccgc aagtggaagt tcaagaagga
     5341 gcgcaacaag ggctacaagc accatgcgga ggacgccctc atcatcgcca acgccgactt
     5401 catcttcaag gagtggaaaa agttggacaa ggccaagaag gtcatggaga accagatgtt
     5461 cgaggagaag caggccgagt ccatgccgga gatcgagaca gagcaggagt acaaggagat
     5521 cttcatcacg ccgcaccaga tcaagcacat caaggacttc aaggactaca agtactccca
     5581 ccgcgtggac aagaagccca accgcgagct catcaacgac accctctact ccacccgcaa
     5641 ggacgacaag ggcaacaccc tcatcgtcaa caacttaaac ggcctctacg acaaggacaa
     5701 cgacaagttg aaaaagttga tcaacaagag ccccgaaaag ttgctcatgt accaccatga
     5761 cccgcagacg taccaaaagt tgaagttgat catggagcag tacggtgacg agaagaaccc
     5821 gctctacaag tactacgagg agaccggcaa ctacctcacc aagtactcca agaaggacaa
     5881 cggcccggtc atcaagaaga tcaagtacta cggcaacaag ttgaatgccc acctggacat
     5941 caccgacgac taccccaact cccgcaacaa agttgtaaag ttgtcgctaa agccgtaccg
     6001 cttcgacgtc tacctggaca acggcgtcta caagttcgtg accgtcaaga acctggacgt
     6061 catcaagaag gagaactact acgaggtcaa ctccaagtgc tacgaggagg ccaaaaagtt
     6121 gaagaagatc tccaaccagg ccgagttcat cgccagcttc tacaacaacg acctcatcaa
     6181 gatcaacggc gagctctacc gcgtcatcgg ggtcaacaac gacctgctga accgcatcga
     6241 ggtcaacatg atcgacatca cctaccgcga gtacctcgag aacatgaacg acaagcgccc
     6301 gcctcgcatc atcaagacca tcgcctccaa gacccagtcc atcaagaagt actccaccga
     6361 catccttggc aacttgtacg aggtcaagag caagaagcac cctcagatca tcaagaaggg
     6421 caagcgccct gctgccacca agaaggctgg ccaggccaag aagaagaagg gatccggggc
     6481 caccaacttt tccctgctga agcaggctgg cgacgtcgag gagaaccctg gccctggctc
     6541 cggaatgtcc agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga
     6601 ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc
     6661 tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat
     6721 gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt
     6781 acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt
     6841 acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt
     6901 tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca
     6961 ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga
     7021 aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca
     7081 ccaaggcgca gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa
     7141 acaacttaaa tgtgaaagtg ggggcagtgg agagggcaga ggaagtctgc taacatgcgg
     7201 tgacgtcgag gagaatcctg gcccaatggc caagcctttg tctcaagaag aatccaccct
     7261 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     7321 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     7381 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     7441 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     7501 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     7561 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     7621 ctgataatct agaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     7681 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     7741 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     7801 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     7861 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     7921 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     7981 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     8041 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     8101 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     8161 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     8221 ttgggccgcc tccccgcagc gctgtcgact ttaagaccaa tgacttacaa ggcagctgta
     8281 gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga
     8341 agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg
     8401 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     8461 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     8521 ttttagtcag tgtggaaaat ctctagcagg gcccgtttaa acccgctgat cagcctcgac
     8581 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     8641 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     8701 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     8761 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     8821 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     8881 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     8941 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     9001 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     9061 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     9121 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     9181 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     9241 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     9301 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     9361 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     9421 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     9481 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     9541 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     9601 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     9661 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     9721 tgaggaacta aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg
     9781 tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg
     9841 acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc
     9901 aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg
     9961 ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg
    10021 agatcggcga gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg
    10081 tgcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat tccaccgccg
    10141 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
    10201 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
    10261 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
    10321 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
    10381 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
    10441 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
    10501 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
    10561 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
    10621 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
    10681 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
    10741 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
    10801 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
    10861 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
    10921 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
    10981 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
    11041 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
    11101 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
    11161 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
    11221 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
    11281 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
    11341 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
    11401 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
    11461 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
    11521 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
    11581 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
    11641 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
    11701 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
    11761 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
    11821 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
    11881 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
    11941 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
    12001 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
    12061 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
    12121 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
    12181 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
    12241 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
    12301 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
    12361 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
    12421 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
    12481 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
    12541 ttccccgaaa agtgccacct gac
//
