LOCUS       sequence-227310-       18320 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Target a Cre-dependent ChrimsonR-tdTomato cassette and a tTA2 
            cassette into the mouse TIGRE locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 18320)
  AUTHORS   Daigle TL, Madisen L, Hage TA, Valley MT, Knoblich U, Larsen RS, 
            Takeno MM, Huang L, Gu H, Larsen R, Mills M, Bosma-Moody A, Siverts 
            LA, Walker M, Graybuck LT, Yao Z, Fong O, Nguyen TN, Garren E, Lenz 
            GH, Chavarha M, Pendergraft J, Harrington J, Hirokawa KE, Harris JA,
            Nicovich PR, McGraw MJ, Ollerenshaw DR, Smith KA, Baker CA, Ting JT,
            Sunkin SM, Lecoq J, Lin MZ, Boyden ES, Murphy GJ, da Costa NM, 
            Waters J, Li L, Tasic B, Zeng H
  TITLE     A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced 
            Brain-Cell-Type Targeting and Functionality.
  JOURNAL   Cell. 2018 Jul 12;174(2):465-480.e22. doi: 
            10.1016/j.cell.2018.06.035.
  PUBMED    30007418
REFERENCE   2  (bases 1 to 18320)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2018.06"; journalName: "Cell"; date: "2018-07-12- 
            12"; volume: "174"; issue: "2"; pages: "465-480.e22"
COMMENT     Sequence Label: Ai167 (TIT2L-ChrimsonR-tdT-ICL-tTA2) targeting...
FEATURES             Location/Qualifiers
     source          1..18320
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2975..3056
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3000..3019
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    join(3070..3088,3089..3111,3112..3130,3131..3153,3154..3172,
                     3173..3195,3196..3214,3215..3237,3238..3256,3257..3279,
                     3280..3298,3299..3321,3322..3340)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        3373..3411
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3408..3432
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3545..3578)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3655..3703
                     /note="synthetic polyadenylation signal"
     polyA_signal    3712..4188
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     polyA_signal    4200..4661
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     protein_bind    complement(4688..4721)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4732..4741
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4738..5787
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     misc_feature    7258..7846
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(7729..7740)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    7877..8084
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        10608..10911
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10913..11190
                     /label=chicken beta-actin promoter
     intron          11191..12207
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     12131..12153
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     12215..12234
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    12295..12328
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     polyA_signal    12366..12414
                     /note="synthetic polyadenylation signal"
     polyA_signal    12423..12899
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(12934..12953)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    12978..13026
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    13205..13238
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     CDS             join(13263..13880,13881..13886,13887..13922,13923..13925,
                     13926..13961,13962..13964,13965..14000,14001..14009)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    14052..14640
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(14523..14534)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    14671..14878
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    14892..14925
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        14952..15451
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(14987..15010)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     15384..15403
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(16099..16115)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(16099..16115)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(16112..16134)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    16123..16139
                     /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(16147..16153,16154..16171,16172..16177))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    16192..16213
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(16330..16347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(16501..17089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(16581..16600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(17260..18051,18052..18120))
                     /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     17883..17902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(18121..18225)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     18293..18311
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accgaagttc
      421 ctattcttca aatagtatag gaacttcttt cggccggcca atcgattgga tccgtttaat
      481 tcgagctcgc tagagcccca tcctcactga ctccgtcctg gagttggatg agagataatg
      541 gccttacgtt gtgccagggg agggtcgggc tggatttagc aagatttacc ttctccaaag
      601 agcggtgctg cagtggcaca gctgcccacg gaggtggggg ggtcaccgtc cctggaggtg
      661 atgaagaact gtggggatgt ggcactgagg gacatggcca gtgggcacgg tgggtgggtt
      721 ggggttggtc ttggggatct tggagggctt ttccagcctt catgatttga cgattgtatg
      781 aacatctaca tggcaattct ccagctgcct gtcccagtcc tactgaccca gctgtatctc
      841 tccaggcaag ctcttccacc ccttctgctt gcatccagac accatcaaac atgcaggctc
      901 agacacaggg accagcagtg tctgtggcct ttttgtgctc ctctccatgc tgggttttaa
      961 cttgctcttt gtccttctat cctatcttct tatccttaag gctgttctga acgctgtgac
     1021 ttggagagtg tcccagagcc ctcaacacct gcatgtccca cgtccatgct gtcctgcact
     1081 tccttatccc caagatctgc ctctccgtga tgcactgaat tggcaaacat gtgtcacccc
     1141 agaccaacaa tgtcacagca aactccccct tgataggaca agggggaatg gctttacact
     1201 gagacagggg aggtttgggt tggatatgag gaggcagttt ttcccccaga gggtggtgac
     1261 gcactgaaca ggttgcccaa ggaggctgtg gatgccccat ccctgcaggc attcaaggcc
     1321 aggctggatg tggctctggg cagcctgggc tgctggttga tgaccctgca catagcaggg
     1381 ggttggatct ggatgagcac tgtgctcctt tgcaacccag gccgttctat gattctgtca
     1441 ttctaaatct ctctttcagc ctaaagcttt ttccccgtat ccccccaggt gtctgcaggc
     1501 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     1561 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     1621 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     1681 ttgggggggg gctgtccctc tagcgagctc ggtacccaga gccccatcct cactgactcc
     1741 gtcctggagt tggatgagag ataatggcct tacgttgtgc caggggaggg tcgggctgga
     1801 tttagcaaga tttaccttct ccaaagagcg gtgctgcagt ggcacagctg cccacggagg
     1861 tgggggggtc accgtccctg gaggtgatga agaactgtgg ggatgtggca ctgagggaca
     1921 tggccagtgg gcacggtggg tgggttgggg ttggtcttgg ggatcttgga gggcttttcc
     1981 agccttcatg atttgacgat tgtatgaaca tctacatggc aattctccag ctgcctgtcc
     2041 cagtcctact gacccagctg tatctctcca ggcaagctct tccacccctt ctgcttgcat
     2101 ccagacacca tcaaacatgc aggctcagac acagggacca gcagtgtctg tggccttttt
     2161 gtgctcctct ccatgctggg ttttaacttg ctctttgtcc ttctatccta tcttcttatc
     2221 cttaaggctg ttctgaacgc tgtgacttgg agagtgtccc agagccctca acacctgcat
     2281 gtcccacgtc catgctgtcc tgcacttcct tatccccaag atctgcctct ccgtgatgca
     2341 ctgaattggc aaacatgtgt caccccagac caacaatgtc acagcaaact cccccttgat
     2401 aggacaaggg ggaatggctt tacactgaga caggggaggt ttgggttgga tatgaggagg
     2461 cagtttttcc cccagagggt ggtgacgcac tgaacaggtt gcccaaggag gctgtggatg
     2521 ccccatccct gcaggcattc aaggccaggc tggatgtggc tctgggcagc ctgggctgct
     2581 ggttgatgac cctgcacata gcagggggtt ggatctggat gagcactgtg ctcctttgca
     2641 acccaggccg ttctatgatt ctgtcattct aaatctctct ttcagcctaa agctttttcc
     2701 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg
     2761 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg
     2821 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga
     2881 gggacgtaat tacatccctg ggggctttgg gggggggctg tccctctagg ggtacccggg
     2941 gatccaccgg tatcgattcg gccggccgac gtcggcagtg aaaaaaatgc tttatttgtg
     3001 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagtttcga
     3061 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca
     3121 gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa
     3181 agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc
     3241 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
     3301 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg
     3361 tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
     3421 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
     3481 gaccgatcca gcctccgcgg ccccgaattc gagctcggta cccggggatc aaattcaatt
     3541 aaatataact tcgtatagca tacattatac gaagttattc gcgatgaata aatgaaagct
     3601 tgcagatccc ctcagcacca tggctagcgg cagcctcgga gtttgaatag atagaataaa
     3661 atatctttat tttcattcca tctgtgtgtt ggttttttgt gtgagatcta cgggtggcat
     3721 ccctgtgacc cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc
     3781 agccttgtcc taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat
     3841 attatggggt ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg
     3901 gcctgcgggg tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca
     3961 atctccgcct cctgggttca agcgattctc ctgcctcagg ctcccgagtt gttgggattc
     4021 caggcatgca tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc
     4081 atattggcca ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc
     4141 aaattgctgg gattacaggc gtgaaccact gctcccttcc ctgtccttct gcctcagcgc
     4201 tgtaagtctg cagaaattga tgatctatta aacaataaag atgtccacta aaatggaagt
     4261 ttttcctgtc atactttgtt aagaagggtg agaacagagt acctacattt tgaatggaag
     4321 gattggagct acgggggtgg gggtggggtg ggattagata aatgcctgct ctttactgaa
     4381 ggctctttac tattgcttta tgataatgtt tcatagttgg atatcataat ttaaacaagc
     4441 aaaaccaaat taagggccag ctcattcctc ccactcatga tctatagatc tatagatctc
     4501 tcgtgggatc attgtttttc tcttgattcc cactttgtgg ttctaagtac tgtggtttcc
     4561 aaatgtgtca gtttcatagc ctgaagaacg agatcagcag cctctgttcc acatacactt
     4621 cattctcagt attgttttgc caagttctaa ttccatcaga aagcttttaa ttaactgaat
     4681 agctaacata acttcgtata gcatacatta tacgaagtta tcacgcgtgc cgccaccatg
     4741 gctgagctga tcagcagcgc caccagatct ctgtttgccg ccggaggcat caacccttgg
     4801 cctaacccct accaccacga ggacatgggc tgtggaggaa tgacacctac aggcgagtgc
     4861 ttcagcaccg agtggtggtg tgacccttct tacggactga gcgacgccgg atacggatat
     4921 tgcttcgtgg aggccacagg cggctacctg gtcgtgggag tggagaagaa gcaggcttgg
     4981 ctgcacagca gaggcacacc aggagaaaag atcggcgccc aggtctgcca gtggattgct
     5041 ttcagcatcg ccatcgccct gctgacattc tacggcttca gcgcctggaa ggccacttgc
     5101 ggttgggagg aggtctacgt ctgttgcgtc gaggtgctgt tcgtgaccct ggagatcttc
     5161 aaggagttca gcagccccgc cacagtgtac ctgtctaccg gcaaccacgc ctattgcctg
     5221 cgctacttcg agtggctgct gtcttgcccc gtgatcctga tcagactgag caacctgagc
     5281 ggcctgaaga acgactacag caagcggacc atgggcctga tcgtgtcttg cgtgggaatg
     5341 atcgtgttcg gcatggccgc aggactggct accgattggc tcaagtggct gctgtatatc
     5401 gtgtcttgca tctacggcgg ctacatgtac ttccaggccg ccaagtgcta cgtggaagcc
     5461 aaccacagcg tgcctaaagg ccattgccgc atggtcgtga agctgatggc ctacgcttac
     5521 ttcgcctctt ggggcagcta cccaatcctc tgggcagtgg gaccagaagg actgctgaag
     5581 ctgagccctt acgccaacag catcggccac agcatctgcg acatcatcgc caaggagttt
     5641 tggaccttcc tggcccacca cctgaggatc aagatccacg agcacatcct gatccacggc
     5701 gacatccgga agaccaccaa gatggagatc ggaggcgagg aggtggaagt ggaagagttc
     5761 gtggaggagg aggacgagga cacagtggcg gcaccggtag tagcagtgag taagggcgag
     5821 gaagtgatca aagagttcat gcggtttaag gtgagaatgg aaggaagcat gaacggccac
     5881 gagttcgaaa ttgagggaga aggagaggga cggccctacg agggcaccca gacagccaag
     5941 ctgaaagtga caaagggcgg gcctctgcca ttcgcttggg acatcctgag cccacagttt
     6001 atgtacggct ccaaggccta tgtgaaacat ccagctgaca ttcccgatta taagaaactg
     6061 agcttccccg aggggtttaa gtgggaaaga gtgatgaact tcgaggacgg aggcctggtg
     6121 actgtgaccc aggacagctc cctgcaggat gggaccctga tctacaaggt gaaaatgaga
     6181 gggacaaatt ttccccctga tggacctgtg atgcagaaga aaactatggg atgggaggcc
     6241 tccaccgaaa ggctgtatcc acgcgacggg gtgctgaaag gagaaatcca ccaggctctg
     6301 aagctgaaag atgggggaca ttacctggtg gagttcaaga caatctacat ggccaagaaa
     6361 cctgtgcagc tgccaggcta ctattacgtg gacacaaaac tggatatcac ttcacacaac
     6421 gaggactaca ctattgtgga gcagtatgaa cggagcgagg ggagacacca tctgttcctg
     6481 ggccatggga ctggaagtac cggctcaggg tctagtggaa ccgcctcaag cgaggataac
     6541 aatatggctg tgatcaaaga gttcatgagg tttaaggtgc gcatggaggg cagcatgaat
     6601 gggcacgaat ttgagattga aggagagggc gaagggaggc cttacgaggg cacacagact
     6661 gccaagctga aagtgaccaa gggaggacca ctgcctttcg cttgggatat cctgtctcct
     6721 cagtttatgt acggaagtaa ggcctatgtc aagcatcccg ctgacattcc tgattacaag
     6781 aaactgtctt tcccagaggg ctttaagtgg gagagagtga tgaattttga agatggaggc
     6841 ctggtgaccg tgacacagga ctcctctctg caggatggca ctctgatcta caaagtcaaa
     6901 atgcgcggca ccaattttcc acccgatggg cccgtgatgc agaagaaaac aatggggtgg
     6961 gaggccagca ctgaacggct gtatcctaga gacggagtgc tgaagggcga aatccaccag
     7021 gccctgaagc tgaaagacgg cggccactac ctggtggagt tcaaaaccat ctacatggcc
     7081 aagaaaccag tgcagctgcc cggctattac tatgtggaca ccaagctgga tatcacatcc
     7141 cacaatgaag actacaccat tgtggaacag tatgagaggt ctgaaggacg ccaccatctg
     7201 tttctgtacg gcatggatga gctgtataag taaacgcgtt acgtaaagct tgtcgataat
     7261 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     7321 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     7381 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     7441 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     7501 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     7561 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     7621 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     7681 tatgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     7741 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     7801 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg acaccgagcg
     7861 ctgctcgagg cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     7921 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     7981 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     8041 agcaaggggg aggattggga agacaatagc aggcatgctg gggacacgtg ggatcctcta
     8101 gagccccatc ctcactgact ccgtcctgga gttggatgag agataatggc cttacgttgt
     8161 gccaggggag ggtcgggctg gatttagcaa gatttacctt ctccaaagag cggtgctgca
     8221 gtggcacagc tgcccacgga ggtggggggg tcaccgtccc tggaggtgat gaagaactgt
     8281 ggggatgtgg cactgaggga catggccagt gggcacggtg ggtgggttgg ggttggtctt
     8341 ggggatcttg gagggctttt ccagccttca tgatttgacg attgtatgaa catctacatg
     8401 gcaattctcc agctgcctgt cccagtccta ctgacccagc tgtatctctc caggcaagct
     8461 cttccacccc ttctgcttgc atccagacac catcaaacat gcaggctcag acacagggac
     8521 cagcagtgtc tgtggccttt ttgtgctcct ctccatgctg ggttttaact tgctctttgt
     8581 ccttctatcc tatcttctta tccttaaggc tgttctgaac gctgtgactt ggagagtgtc
     8641 ccagagccct caacacctgc atgtcccacg tccatgctgt cctgcacttc cttatcccca
     8701 agatctgcct ctccgtgatg cactgaattg gcaaacatgt gtcaccccag accaacaatg
     8761 tcacagcaaa ctcccccttg ataggacaag ggggaatggc tttacactga gacaggggag
     8821 gtttgggttg gatatgagga ggcagttttt cccccagagg gtggtgacgc actgaacagg
     8881 ttgcccaagg aggctgtgga tgccccatcc ctgcaggcat tcaaggccag gctggatgtg
     8941 gctctgggca gcctgggctg ctggttgatg accctgcaca tagcaggggg ttggatctgg
     9001 atgagcactg tgctcctttg caacccaggc cgttctatga ttctgtcatt ctaaatctct
     9061 ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
     9121 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
     9181 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
     9241 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
     9301 tgtccctcta gagccccatc ctcactgact ccgtcctgga gttggatgag agataatggc
     9361 cttacgttgt gccaggggag ggtcgggctg gatttagcaa gatttacctt ctccaaagag
     9421 cggtgctgca gtggcacagc tgcccacgga ggtggggggg tcaccgtccc tggaggtgat
     9481 gaagaactgt ggggatgtgg cactgaggga catggccagt gggcacggtg ggtgggttgg
     9541 ggttggtctt ggggatcttg gagggctttt ccagccttca tgatttgacg attgtatgaa
     9601 catctacatg gcaattctcc agctgcctgt cccagtccta ctgacccagc tgtatctctc
     9661 caggcaagct cttccacccc ttctgcttgc atccagacac catcaaacat gcaggctcag
     9721 acacagggac cagcagtgtc tgtggccttt ttgtgctcct ctccatgctg ggttttaact
     9781 tgctctttgt ccttctatcc tatcttctta tccttaaggc tgttctgaac gctgtgactt
     9841 ggagagtgtc ccagagccct caacacctgc atgtcccacg tccatgctgt cctgcacttc
     9901 cttatcccca agatctgcct ctccgtgatg cactgaattg gcaaacatgt gtcaccccag
     9961 accaacaatg tcacagcaaa ctcccccttg ataggacaag ggggaatggc tttacactga
    10021 gacaggggag gtttgggttg gatatgagga ggcagttttt cccccagagg gtggtgacgc
    10081 actgaacagg ttgcccaagg aggctgtgga tgccccatcc ctgcaggcat tcaaggccag
    10141 gctggatgtg gctctgggca gcctgggctg ctggttgatg accctgcaca tagcaggggg
    10201 ttggatctgg atgagcactg tgctcctttg caacccaggc cgttctatga ttctgtcatt
    10261 ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt ctgcaggctc
    10321 aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg
    10381 ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc
    10441 cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt
    10501 gggggggggc tgtccctcta gagtcgacgg cgcgccctgc aggaatctag ttattaatag
    10561 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
    10621 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
    10681 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
    10741 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
    10801 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
    10861 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg
    10921 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat
    10981 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg
    11041 ccrggsgggg sggggsgggg cgrggggsgg ggcggggcga ggcggagagg tgcggcggca
    11101 gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg
    11161 ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg cgcgctgcct tcgccccgtg
    11221 ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc gttactccca
    11281 caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt ggtttaatga
    11341 cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga gggccctttg
    11401 tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag cgccgcgtgc
    11461 ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct ttgtgcgctc
    11521 cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg ggggggctgc
    11581 gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg gggtgtgggc
    11641 gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga gcacggcccg
    11701 gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc gggcgggggg
    11761 tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga gggctcgggg
    11821 gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag ccgcagccat
    11881 tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca aatctgtgcg
    11941 gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc gaagcggtgc
    12001 ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg ccgccgtccc
    12061 cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg ggggggacgg
    12121 ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc tgctaaccat
    12181 gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat tgtgctgtct
    12241 catcattttg gcaaagaatt gatttgatac cgcgggcccc gcgtggacgg taccataact
    12301 tcgtataaag tatcctatac gaagttatcc atggctagcg gcagcctcgg agtttgaata
    12361 gatagaataa aatatcttta ttttcattcc atctgtgtgt tggttttttg tgtgagatct
    12421 acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc
    12481 cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt
    12541 ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga
    12601 caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt
    12661 ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag gctcccgagt
    12721 tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac
    12781 ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac
    12841 cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtccttc
    12901 tgcctcagct aattgagtag gggggaggct aactgaaaca cggaaggaga caataccgga
    12961 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt
    13021 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
    13081 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
    13141 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccgtggaagt tcctattctc
    13201 tagaataact tcgtataaag tatcctatac gaagttatac gcgcgcgtcc atttaattca
    13261 ccatgtctag actggacaag agcaaagtca taaactctgc tctggaatta ctcaatgaag
    13321 tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt gagcagccta
    13381 ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctggca atcgagatgc
    13441 tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa gactttctgc
    13501 ggaacaacgc caagtcattc cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc
    13561 atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc
    13621 tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc gtgggccact
    13681 ttacactggg ctgcgtattg gaggatcagg agcatcaagt agcaaaagag gaaagagaga
    13741 cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg ttcgaccatc
    13801 agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc ctggagaaac
    13861 agctaaagtg cgaaagcggc gggccggccg acgcccttga cgattttgac ttagacatgc
    13921 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
    13981 attttgacct tgacatgctc cccgggtaac taagtaagga tcaaatggcg cgttacgtaa
    14041 agcttgtcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
    14101 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
    14161 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
    14221 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
    14281 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
    14341 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
    14401 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
    14461 cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
    14521 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
    14581 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
    14641 atcgacaccg agcgctgctc gaggcctcga ctgtgccttc tagttgccag ccatctgttg
    14701 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
    14761 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
    14821 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggaca
    14881 cgtgggatcc ggtgccaggg cgtgcccttg ggctccccgg gcgcgtttgg gcggccgcat
    14941 cgaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc
    15001 agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc
    15061 caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc
    15121 tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat
    15181 ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg
    15241 ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag
    15301 gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc
    15361 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
    15421 gttctcctct tcctcatctc cgggcctttc gacctgcaat cgccgctagc cgccaccatg
    15481 aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc
    15541 gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta
    15601 ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt
    15661 tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg
    15721 gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa
    15781 gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg
    15841 atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc
    15901 ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac
    15961 tggcaaactg tgatggacga caccgtcagt gtaagtgcta gcccctaggc ctgaatcgaa
    16021 gttcctattc ttcaaaaggt ataggaactt cgagctcgtc gactgcagag gcctgcatgc
    16081 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
    16141 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
    16201 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
    16261 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
    16321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
    16381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
    16441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
    16501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
    16561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
    16621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
    16681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
    16741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
    16801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
    16861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
    16921 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
    16981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
    17041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
    17101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
    17161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
    17221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
    17281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
    17341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
    17401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
    17461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
    17521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
    17581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
    17641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
    17701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
    17761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
    17821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
    17881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
    17941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
    18001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
    18061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
    18121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
    18181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
    18241 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
    18301 tatcacgagg ccctttcgtc
//
