LOCUS       sequence-390328-       12574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA targeting chinise hamster Bip (Hspa5) with CRISPRa 
            components and mCherry, blasticidin selective .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12574)
  AUTHORS   Melo EP, Konno T, Farace I, Awadelkareem MA, Skov LR, Teodoro F, 
            Sancho TP, Paton AW, Paton JC, Fares M, Paulo PMR, Zhang X, Avezov E
  TITLE     Stress-induced protein disaggregation in the endoplasmic reticulum 
            catalysed by BiP.
  JOURNAL   Nat Commun. 2022 May 6;13(1):2501. doi: 10.1038/s41467-022-30238-2.
  PUBMED    35523806
REFERENCE   2  (bases 1 to 12574)
  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: "2022-05-6"; volume: "13(1):2501. doi"; pages: " 
            10.1038/s41467-022-30238-2"
COMMENT     Sequence Label: pCRISPRa_all-in-one_Bip gRNA1
FEATURES             Location/Qualifiers
     source          1..12574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        287..305
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        357..375
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        554..1732
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          785..1723
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1680..1700
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1739..1748
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1748..2134
                     /codon_start=1
                     /product="bacteriophage MS2 coat protein"
                     /label=MS2-N55K
                     /note="binds with high affinity to a specific stem-loop 
                     structure in the viral RNA (Lim et al., 1994)"
                     /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT
                     CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI
                     VKAMQGLLKDGNPIPSAIAANSGIY"
     CDS             2189..2209
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2225..2767
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2792..3163
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             3179..3232
                     /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             3233..3937
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3375..3393)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3643..3662
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3947..4000
                     /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             4001..4048
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(4049..4072,4073..4075,4076..6631,6632..6634,
                     6635..8149)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the N863A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             8204..8224
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8249..8398
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             8420..8473
                     /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             8474..8872
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(9216..9233)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9222..9446
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   9733..9767
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(9787..9807)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(9787..9805)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(9826..9842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9826..9842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9839..9861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9850..9866
                     /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(9874..9880,9881..9898,9899..9904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9919..9940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(10057..10074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(10228..10816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(10308..10327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10987..11778,11779..11847))
                     /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     11610..11629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11848..11952)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(11978..12433)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(12115..12136)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     12327..12346
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg tggtggcatg gaccaatcag ttttagagct aggccaacat gaggatcacc
      301 catgtctgca gggcctagca agttaaaata aggctagtcc gttatcaact tggccaacat
      361 gaggatcacc catgtctgca gggccaagtg gcaccgagtc ggtgcttttt ttgctttgtt
      421 actttataga agaaattttg agtttttgtt tttttttaat aaataaataa acataaataa
      481 attgtttgtt gaatttatta ttagtatgta agtgtaaata taataaaact taatatctat
      541 tcaaattaat aaaggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
      601 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
      661 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
      721 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
      781 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
      841 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
      901 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
      961 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
     1021 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
     1081 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
     1141 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
     1201 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
     1261 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
     1321 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     1381 ctcaaaatga aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     1441 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     1501 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     1561 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     1621 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     1681 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gacgtacggc
     1741 caccatggct tcaaacttta ctcagttcgt gctcgtggac aatggtggga caggggatgt
     1801 gacagtggct ccttctaatt tcgctaatgg ggtggcagag tggatcagct ccaactcacg
     1861 gagccaggcc tacaaggtga catgcagcgt caggcagtct agtgcccaga agagaaagta
     1921 taccatcaag gtggaggtcc ccaaagtggc tacccagaca gtgggcggag tcgaactgcc
     1981 tgtcgccgct tggaggtcct acctgaacat ggagctcact atcccaattt tcgctaccaa
     2041 ttctgactgt gaactcatcg tgaaggcaat gcaggggctc ctcaaagacg gtaatcctat
     2101 cccttccgcc atcgccgcta actcaggtat ctacagcgct ggaggaggtg gaagcggagg
     2161 aggaggaagc ggaggaggag gtagcggacc taagaaaaag aggaaggtgg cggccgctgg
     2221 atccccttca gggcagatca gcaaccaggc cctggctctg gcccctagct ccgctccagt
     2281 gctggcccag actatggtgc cctctagtgc tatggtgcct ctggcccagc cacctgctcc
     2341 agcccctgtg ctgaccccag gaccacccca gtcactgagc gctccagtgc ccaagtctac
     2401 acaggccggc gaggggactc tgagtgaagc tctgctgcac ctgcagttcg acgctgatga
     2461 ggacctggga gctctgctgg ggaacagcac cgatcccgga gtgttcacag atctggcctc
     2521 cgtggacaac tctgagtttc agcagctgct gaatcagggc gtgtccatgt ctcatagtac
     2581 agccgaacca atgctgatgg agtaccccga agccattacc cggctggtga ccggcagcca
     2641 gcggcccccc gaccccgctc caactcccct gggaaccagc ggcctgccta atgggctgtc
     2701 cggagatgaa gacttctcaa gcatcgctga tatggacttt agtgccctgc tgtcacagat
     2761 ttcctctagt gggcagggag gaggtggaag cggcttcagc gtggacacca gtgccctgct
     2821 ggacctgttc agcccctcgg tgaccgtgcc cgacatgagc ctgcctgacc ttgacagcag
     2881 cctggccagt atccaagagc tcctgtctcc ccaggagccc cccaggcctc ccgaggcaga
     2941 gaacagcagc ccggattcag ggaagcagct ggtgcactac acagcgcagc cgctgttcct
     3001 gctggacccc ggctccgtgg acaccgggag caacgacctg ccggtgctgt ttgagctggg
     3061 agagggctcc tacttctccg aaggggacgg cttcgccgag gaccccacca tctccctgct
     3121 gacaggctcg gagcctccca aagccaagga ccccactgtc tcctgtacag gcagtggaga
     3181 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa
     3241 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     3301 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     3361 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     3421 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     3481 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     3541 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     3601 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     3661 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     3721 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     3781 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     3841 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     3901 ccgccactcc accggcggca tggacgagct gtacaaggga tccggagagg gcagaggaag
     3961 tctcctaaca tgcggtgacg tggaggagaa tcctggccca aaaaggccgg cggccacgaa
     4021 aaaggccggc caggcaaaaa agaaaaagga caagaagtac agcatcggcc tggccatcgg
     4081 caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt
     4141 caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct
     4201 gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata
     4261 caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa
     4321 ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa
     4381 gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta
     4441 ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg
     4501 gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg
     4561 cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta
     4621 caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct
     4681 gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga
     4741 gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga cccccaactt
     4801 caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga
     4861 cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc
     4921 cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat
     4981 caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct
     5041 gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt
     5101 cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt
     5161 ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa
     5221 gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca
     5281 ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt
     5341 cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt
     5401 gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac
     5461 catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat
     5521 cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag
     5581 cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga
     5641 gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct
     5701 gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat
     5761 cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg
     5821 cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa
     5881 cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat
     5941 cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa
     6001 gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga
     6061 caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa
     6121 cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca
     6181 ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc
     6241 cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg
     6301 ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa
     6361 gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg
     6421 cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta
     6481 cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg
     6541 gctgtccgac tacgatgtgg accacatcgt gcctcagagc tttctgaagg acgactccat
     6601 cgacaacaag gtgctgacca gaagcgacaa ggcccggggc aagagcgaca acgtgccctc
     6661 cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat
     6721 tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga
     6781 taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc
     6841 acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga
     6901 agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt
     6961 ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt
     7021 cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga
     7081 ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc
     7141 taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct
     7201 ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat
     7261 cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt
     7321 gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc
     7381 caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg
     7441 cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg
     7501 caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag
     7561 cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa
     7621 ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag
     7681 aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata
     7741 tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa
     7801 tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca
     7861 gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc
     7921 cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct
     7981 gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga
     8041 ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat
     8101 caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca gcgctggagg
     8161 aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga aaaagaggaa
     8221 ggtggcggcc gctggatccg gacgggctga cgcattggac gattttgatc tggatatgct
     8281 gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga
     8341 ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat
     8401 taactgtaca ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
     8461 gaatcctggc ccaatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     8521 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     8581 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     8641 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     8701 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     8761 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     8821 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct aagtaccttt
     8881 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     8941 actggaaggg ctaattcact cccaaagaag tcaagatctg ctttttgcct gtactgggtc
     9001 tctctggtta gaccagagtc tctctggtta gaccagatct gagcctggga gctctctggc
     9061 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     9121 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     9181 tggaaaatct ctagcagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     9241 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     9301 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     9361 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca atagcaggca
     9421 tgctggggat gcggtgggct ctatgggcgg ccgttaatga tatctataac aagaaaatat
     9481 atatataata agttatcacg taagtagaac atgaaataac aatataatta tcgtatgagt
     9541 taaatcttaa aagtcacgta aaagataatc atgcgtcatt ttgactcacg cggtcgttat
     9601 agttcaaaat cagtgacact taccgcattg acaagcacgc ctcacgggag ctccaagcgg
     9661 cgactgagat gtcctaaatg cacagcgacg gattcgcgct atttagaaag agagagcaat
     9721 atttcaagaa tgcatgcgtc aattttacgc agactatctt tctagggtta aattaagctt
     9781 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     9841 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg
     9901 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     9961 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
    10021 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
    10081 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
    10141 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
    10201 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
    10261 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
    10321 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
    10381 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
    10441 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
    10501 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
    10561 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
    10621 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
    10681 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
    10741 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
    10801 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
    10861 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
    10921 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
    10981 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
    11041 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
    11101 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
    11161 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
    11221 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
    11281 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
    11341 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
    11401 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
    11461 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
    11521 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
    11581 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
    11641 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
    11701 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
    11761 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
    11821 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
    11881 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
    11941 aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg
    12001 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
    12061 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
    12121 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
    12181 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
    12241 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
    12301 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
    12361 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
    12421 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
    12481 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
    12541 tgggtaacgc cagggttttc ccagtcacga cgtt
//
