LOCUS       sequence-432498-       13023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting human SPNS1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13023)
  AUTHORS   Kenny TC, Khan A, Son Y, Yue L, Heissel S, Sharma A, Pasolli HA, Liu
            Y, Gamazon ER, Alwaseem H, Hite RK, Birsoy K
  TITLE     Integrative genetic analysis identifies FLVCR1 as a plasma-membrane 
            choline transporter in mammals.
  JOURNAL   Cell Metab. 2023 Jun 6;35(6):1057-1071.e12. doi: 
            10.1016/j.cmet.2023.04.003. Epub 2023 Apr 25.
  PUBMED    37100056
REFERENCE   2  (bases 1 to 13023)
  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"; doi: 
            "10.1016/j.cmet.2023.04.003"; journalName: "Cell Metab"; date: 
            "2023-06-6- 6"; volume: "35"; issue: "6"; pages: "1057-1071.e12"
COMMENT     Sequence Label: plentiCRISPR-v2 Puro SPNS1_sg1
FEATURES             Location/Qualifiers
     source          1..13023
                     /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        2608..2856
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2608..2628
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2779..2798
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3025..3236
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3255..3264
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3261..7364
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3261..7364
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7365..7412
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7413..7436
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             7446..7502
                     /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             7503..8099
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     7996..8016
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    8115..8703
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(8168..8188)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(8586..8597)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             8775..9008
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(9034..9051)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    9040..9264
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      9310..9738
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9397..9416)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9607..9628
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(9747..9767)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9752..10081
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9932..10067
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9994..10013
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        10129..10176
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             10195..10569
                     /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    10699..10820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10736..10755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10790..10809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(10869..10885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(10869..10885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(10882..10904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    10893..10909
                     /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(10917..10923,10924..10941,10942..10947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10962..10983
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(11100..11117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(11271..11859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(11351..11370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(12030..12821,12822..12890))
                     /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     12653..12672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(12891..12995)
                     /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 aggtaccgag ggcctatttc ccatgattcc ttcatatttg
     2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
     2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2821 ttggctttat atatcttgtg gaaaggacga aacaccgcta catggaccgc ttcaccggtt
     2881 taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg
     2941 aaaaagtggc accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg
     3001 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     3061 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
     3121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
     3241 ggttctagag cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca
     3301 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
     3361 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
     3421 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
     3481 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
     3541 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
     3601 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
     3661 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
     3721 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
     3781 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
     3841 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
     3901 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
     3961 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
     4021 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
     4081 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     4141 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     4201 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     4261 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     4321 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     4381 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     4441 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     4501 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     4561 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     4621 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     4681 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     4741 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     4801 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     4861 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     4921 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     4981 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     5041 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     5101 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     5161 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     5221 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     5281 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     5341 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     5401 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     5461 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     5521 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     5581 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     5641 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     5701 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     5761 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     5821 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     5881 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     5941 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     6001 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     6061 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     6121 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     6181 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     6241 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     6301 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     6361 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     6421 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     6481 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     6541 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     6601 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     6661 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     6721 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     6781 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     6841 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     6901 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     6961 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     7021 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     7081 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     7141 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     7201 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     7261 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     7321 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca
     7381 caaagaaggc tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat
     7441 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     7501 cgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     7561 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     7621 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     7681 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     7741 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     7801 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     7861 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     7921 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     7981 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     8041 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     8101 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     8161 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     8221 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     8281 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     8341 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     8401 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     8461 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     8521 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     8581 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     8641 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     8701 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     8761 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     8821 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     8881 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     8941 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     9001 ctctagcagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     9061 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     9121 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     9181 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     9241 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     9301 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     9361 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     9421 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     9481 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     9541 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     9601 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     9661 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     9721 aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     9781 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     9841 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     9901 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     9961 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
    10021 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
    10081 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa
    10141 tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc
    10201 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
    10261 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
    10321 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
    10381 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
    10441 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
    10501 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
    10561 caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
    10621 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
    10681 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
    10741 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
    10801 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
    10861 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
    10921 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
    10981 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
    11041 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
    11101 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
    11161 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
    11221 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
    11281 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
    11341 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
    11401 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
    11461 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
    11521 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
    11581 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
    11641 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
    11701 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
    11761 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
    11821 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
    11881 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
    11941 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
    12001 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
    12061 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
    12121 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
    12181 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
    12241 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
    12301 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
    12361 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
    12421 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
    12481 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
    12541 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
    12601 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
    12661 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
    12721 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
    12781 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
    12841 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
    12901 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
    12961 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
    13021 gac
//
