LOCUS       sequence-221183-       17200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Target a Cre-dependent GCaMP6f expression 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 17200)
  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 17200)
  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.cell.2018.06"; journalName: "Cell"; date: "2018-07-12- 
            12"; volume: "174"; issue: "2"; pages: "465-480.e22"
COMMENT     Sequence Label: Ai148 (TIT2L-GC6f-ICL-tTA2) targeting vector
FEATURES             Location/Qualifiers
     source          1..17200
                     /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"
     protein_bind    414..447
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     polyA_signal    2959..3040
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2984..3003
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    join(3054..3072,3073..3095,3096..3114,3115..3137,3138..3156,
                     3157..3179,3180..3198,3199..3221,3222..3240,3241..3263,
                     3264..3282,3283..3305,3306..3324)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        3357..3395
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3392..3416
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    complement(3529..3562)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3640..3688
                     /note="synthetic polyadenylation signal"
     polyA_signal    3697..4173
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     polyA_signal    4185..4646
                     /label=PGK poly(A) signal
                     /note="mouse phosphoglycerate kinase 1 polyadenylation 
                     signal"
     protein_bind    complement(4673..4706)
                     /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             join(4738..4746,4747..4764,4765..4767,4768..4800,4801..4803,
                     4804..4827,4828..4854,4855..4911,4912..4917,4918..5187,
                     5188..5205,5206..5640,5641..5646,5647..6090)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      4834..4843
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5124..5145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5251..5272)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5512..5531)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    6133..6721
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(6604..6615)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6752..6959
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        9490..9793
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        9795..10072
                     /label=chicken beta-actin promoter
     intron          10074..11091
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     11015..11037
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     11099..11118
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    11179..11212
                     /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    11250..11298
                     /note="synthetic polyadenylation signal"
     polyA_signal    11307..11783
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(11818..11837)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    11862..11910
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     protein_bind    12089..12122
                     /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(12143..12760,12761..12766,12767..12802,12803..12805,
                     12806..12841,12842..12844,12845..12880,12881..12889)
                     /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    12932..13520
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(13403..13414)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    13551..13758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    13772..13805
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     promoter        13832..14331
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(13867..13890)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     14264..14283
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     complement(14979..14995)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(14979..14995)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(14992..15014)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    15003..15019
                     /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(15027..15033,15034..15051,15052..15057))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    15072..15093
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(15210..15227)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(15381..15969)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(15461..15480)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(16140..16931,16932..17000))
                     /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     16763..16782
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(17001..17105)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     17173..17191
                     /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 gctgtcccga gctcggtacc gccccatcct cactgactcc gtcctggagt
     1741 tggatgagag ataatggcct tacgttgtgc caggggaggg tcgggctgga tttagcaaga
     1801 tttaccttct ccaaagagcg gtgctgcagt ggcacagctg cccacggagg tgggggggtc
     1861 accgtccctg gaggtgatga agaactgtgg ggatgtggca ctgagggaca tggccagtgg
     1921 gcacggtggg tgggttgggg ttggtcttgg ggatcttgga gggcttttcc agccttcatg
     1981 atttgacgat tgtatgaaca tctacatggc aattctccag ctgcctgtcc cagtcctact
     2041 gacccagctg tatctctcca ggcaagctct tccacccctt ctgcttgcat ccagacacca
     2101 tcaaacatgc aggctcagac acagggacca gcagtgtctg tggccttttt gtgctcctct
     2161 ccatgctggg ttttaacttg ctctttgtcc ttctatccta tcttcttatc cttaaggctg
     2221 ttctgaacgc tgtgacttgg agagtgtccc agagccctca acacctgcat gtcccacgtc
     2281 catgctgtcc tgcacttcct tatccccaag atctgcctct ccgtgatgca ctgaattggc
     2341 aaacatgtgt caccccagac caacaatgtc acagcaaact cccccttgat aggacaaggg
     2401 ggaatggctt tacactgaga caggggaggt ttgggttgga tatgaggagg cagtttttcc
     2461 cccagagggt ggtgacgcac tgaacaggtt gcccaaggag gctgtggatg ccccatccct
     2521 gcaggcattc aaggccaggc tggatgtggc tctgggcagc ctgggctgct ggttgatgac
     2581 cctgcacata gcagggggtt ggatctggat gagcactgtg ctcctttgca acccaggccg
     2641 ttctatgatt ctgtcattct aaatctctct ttcagcctaa agctttttcc ccgtatcccc
     2701 ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc
     2761 accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc
     2821 cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat
     2881 tacatccctg ggggctttgg gggggggctg tcccggtacc cggggatcca ccggtatcga
     2941 ttcggccggc cgacgtcggc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     3001 tgctttattt gtaaccatta taagctgcaa taaacaagtt tcgagtttac cactccctat
     3061 cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata gagaaaagtg
     3121 aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga gtttaccact
     3181 ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga
     3241 aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt
     3301 accactccct atcagtgata gagaaaagtg aaagtcgagc tcggtacccg ggtcgaggta
     3361 ggcgtgtacg gtgggaggcc tatataagca gagctcgttt agtgaaccgt cagatcgcct
     3421 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
     3481 gcggccccga attcgagctc ggtacccggg gatcaaattc aattaaatat aacttcgtat
     3541 agcatacatt atacgaagtt attcgcgagt gaataaatga aagcttgcag atcccctcag
     3601 caccatggct agcggcagcc tcggagtttg aatagataga ataaaatatc tttattttca
     3661 ttccatctgt gtgttggttt tttgtgtgag atctacgggt ggcatccctg tgacccctcc
     3721 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata
     3781 aaattaagtt gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg
     3841 ggggtggtat ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat
     3901 tgggaaccaa gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg
     3961 gttcaagcga ttctcctgcc tcaggctccc gagttgttgg gattccaggc atgcatgacc
     4021 aggctcagct aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg
     4081 gtctccaact cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta
     4141 caggcgtgaa ccactgctcc cttccctgtc cttctgcctc agcgctgtaa gtctgcagaa
     4201 attgatgatc tattaaacaa taaagatgtc cactaaaatg gaagtttttc ctgtcatact
     4261 ttgttaagaa gggtgagaac agagtaccta cattttgaat ggaaggattg gagctacggg
     4321 ggtgggggtg gggtgggatt agataaatgc ctgctcttta ctgaaggctc tttactattg
     4381 ctttatgata atgtttcata gttggatatc ataatttaaa caagcaaaac caaattaagg
     4441 gccagctcat tcctcccact catgatctat agatctatag atctctcgtg ggatcattgt
     4501 ttttctcttg attcccactt tgtggttcta agtactgtgg tttccaaatg tgtcagtttc
     4561 atagcctgaa gaacgagatc agcagcctct gttccacata cacttcattc tcagtattgt
     4621 tttgccaagt tctaattcca tcagaaagct tttaattaac tgaatagcta acataacttc
     4681 gtatagcata cattatacga agttatcacg cgccatttgg gaaccggtgc cgccaccatg
     4741 ggttctcatc atcatcatca tcatggtatg gctagcatga ctggtggaca gcaaatgggt
     4801 cgggatctgt acgacgatga cgataaggat ctcgccacca tggtcgactc atcacgtcgt
     4861 aagtggaata agacaggtca cgcagtcaga gctataggtc ggctgagctc actcgagaac
     4921 gtctatatca aggccgacaa gcagaagaac ggcatcaagg cgaacttcaa gatccgccac
     4981 aacatcgagg acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc
     5041 gacggccccg tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actttcgaaa
     5101 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     5161 actctcggca tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc
     5221 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     5281 cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa gctgaccctg
     5341 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     5401 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     5461 aagtccgcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc
     5521 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     5581 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     5641 ctgccggacc aactgactga agagcagatc gcagaattta aagaggaatt ctccctattt
     5701 gacaaggacg gggatgggac aataacaacc aaggagctgg ggacggtgat gcggtctctg
     5761 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt
     5821 gacggcacaa tcgacttccc tgagttcctg acaatgatgg caagaaaaat gaaatacagg
     5881 gacacggaag aagaaattag agaagcgttc ggtgtgtttg ataaggatgg caatggctac
     5941 atcagtgcag cagagcttcg ccacgtgatg acaaaccttg gagagaagtt aacagatgaa
     6001 gaggttgatg aaatgatcag ggaagcagac atcgatgggg atggtcaggt aaactacgaa
     6061 gagtttgtac aaatgatgac agcgaagtaa ggtgtacaaa gataaatggc gcgttacgta
     6121 aagcttgtcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     6181 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     6241 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     6301 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     6361 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     6421 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     6481 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     6541 ccttggctgc tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     6601 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     6661 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     6721 catcgacacc gagcgctgct cgaggcctcg actgtgcctt ctagttgcca gccatctgtt
     6781 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     6841 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     6901 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggac
     6961 acgtgggatc ctctagtcta gagccccatc ctcactgact ccgtcctgga gttggatgag
     7021 agataatggc cttacgttgt gccaggggag ggtcgggctg gatttagcaa gatttacctt
     7081 ctccaaagag cggtgctgca gtggcacagc tgcccacgga ggtggggggg tcaccgtccc
     7141 tggaggtgat gaagaactgt ggggatgtgg cactgaggga catggccagt gggcacggtg
     7201 ggtgggttgg ggttggtctt ggggatcttg gagggctttt ccagccttca tgatttgacg
     7261 attgtatgaa catctacatg gcaattctcc agctgcctgt cccagtccta ctgacccagc
     7321 tgtatctctc caggcaagct cttccacccc ttctgcttgc atccagacac catcaaacat
     7381 gcaggctcag acacagggac cagcagtgtc tgtggccttt ttgtgctcct ctccatgctg
     7441 ggttttaact tgctctttgt ccttctatcc tatcttctta tccttaaggc tgttctgaac
     7501 gctgtgactt ggagagtgtc ccagagccct caacacctgc atgtcccacg tccatgctgt
     7561 cctgcacttc cttatcccca agatctgcct ctccgtgatg cactgaattg gcaaacatgt
     7621 gtcaccccag accaacaatg tcacagcaaa ctcccccttg ataggacaag ggggaatggc
     7681 tttacactga gacaggggag gtttgggttg gatatgagga ggcagttttt cccccagagg
     7741 gtggtgacgc actgaacagg ttgcccaagg aggctgtgga tgccccatcc ctgcaggcat
     7801 tcaaggccag gctggatgtg gctctgggca gcctgggctg ctggttgatg accctgcaca
     7861 tagcaggggg ttggatctgg atgagcactg tgctcctttg caacccaggc cgttctatga
     7921 ttctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
     7981 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
     8041 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     8101 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     8161 tgggggcttt gggggggggc tgtccctcta gagccccatc ctcactgact ccgtcctgga
     8221 gttggatgag agataatggc cttacgttgt gccaggggag ggtcgggctg gatttagcaa
     8281 gatttacctt ctccaaagag cggtgctgca gtggcacagc tgcccacgga ggtggggggg
     8341 tcaccgtccc tggaggtgat gaagaactgt ggggatgtgg cactgaggga catggccagt
     8401 gggcacggtg ggtgggttgg ggttggtctt ggggatcttg gagggctttt ccagccttca
     8461 tgatttgacg attgtatgaa catctacatg gcaattctcc agctgcctgt cccagtccta
     8521 ctgacccagc tgtatctctc caggcaagct cttccacccc ttctgcttgc atccagacac
     8581 catcaaacat gcaggctcag acacagggac cagcagtgtc tgtggccttt ttgtgctcct
     8641 ctccatgctg ggttttaact tgctctttgt ccttctatcc tatcttctta tccttaaggc
     8701 tgttctgaac gctgtgactt ggagagtgtc ccagagccct caacacctgc atgtcccacg
     8761 tccatgctgt cctgcacttc cttatcccca agatctgcct ctccgtgatg cactgaattg
     8821 gcaaacatgt gtcaccccag accaacaatg tcacagcaaa ctcccccttg ataggacaag
     8881 ggggaatggc tttacactga gacaggggag gtttgggttg gatatgagga ggcagttttt
     8941 cccccagagg gtggtgacgc actgaacagg ttgcccaagg aggctgtgga tgccccatcc
     9001 ctgcaggcat tcaaggccag gctggatgtg gctctgggca gcctgggctg ctggttgatg
     9061 accctgcaca tagcaggggg ttggatctgg atgagcactg tgctcctttg caacccaggc
     9121 cgttctatga ttctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc
     9181 ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg
     9241 ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc
     9301 gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta
     9361 attacatccc tgggggcttt gggggggggc tgtccctcta gaagtcgacg ggcgcgccct
     9421 gcaggaatct agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     9481 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     9541 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     9601 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     9661 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     9721 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     9781 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc
     9841 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc
     9901 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc
     9961 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat
    10021 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cggggagtcg
    10081 ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg
    10141 ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc
    10201 tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt
    10261 gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt
    10321 gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg
    10381 cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt
    10441 gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg
    10501 ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct
    10561 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg
    10621 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg
    10681 cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt
    10741 cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga
    10801 cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct
    10861 agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
    10921 cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg
    10981 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
    11041 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
    11101 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgatttg ataccgcggg
    11161 ccccgcgtgg acggtaccat aacttcgtat aaagtatcct atacgaagtt atccatggct
    11221 agcggcagcc tcggagtttg aatagataga ataaaatatc tttattttca ttccatctgt
    11281 gtgttggttt tttgtgtgag atctacgggt ggcatccctg tgacccctcc ccagtgcctc
    11341 tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt
    11401 gcatcatttt gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat
    11461 ggagcaaggg gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa
    11521 gctggagtgc agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga
    11581 ttctcctgcc tcaggctccc gagttgttgg gattccaggc atgcatgacc aggctcagct
    11641 aatttttgtt tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact
    11701 cctaatctca ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa
    11761 ccactgctcc cttccctgtc cttctgcctc agctaattga gtagggggga ggctaactga
    11821 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
    11881 taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
    11941 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
    12001 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
    12061 caggccgtgg aagttcctat tctctagaat aacttcgtat aaagtatcct atacgaagtt
    12121 atacgcgtcc atttaattca ccatgtctag actggacaag agcaaagtca taaactctgc
    12181 tctggaatta ctcaatgaag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa
    12241 gctgggagtt gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga
    12301 tgccctggca atcgagatgc tggacaggca tcatacccac ttctgccccc tggaaggcga
    12361 gtcatggcaa gactttctgc ggaacaacgc caagtcattc cgctgtgctc tcctctcaca
    12421 tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct
    12481 ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc
    12541 tctgtccgcc gtgggccact ttacactggg ctgcgtattg gaggatcagg agcatcaagt
    12601 agcaaaagag gaaagagaga cacctaccac cgattctatg cccccacttc tgagacaagc
    12661 aattgagctg ttcgaccatc agggagccga acctgccttc cttttcggcc tggaactaat
    12721 catatgtggc ctggagaaac agctaaagtg cgaaagcggc gggccggccg acgcccttga
    12781 cgattttgac ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct
    12841 gcctgctgac gctcttgacg attttgacct tgacatgctc cccgggtaac taagtaagga
    12901 tcaaatggcg cgttacgtaa agcttgtcga taatcaacct ctggattaca aaatttgtga
    12961 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
    13021 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
    13081 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
    13141 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
    13201 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
    13261 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
    13321 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
    13381 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
    13441 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
    13501 cctttgggcc gcctccccgc atcgacaccg agcgctgctc gaggcctcga ctgtgccttc
    13561 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
    13621 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
    13681 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
    13741 tagcaggcat gctggggaca cgtgggatcc ggtgccaggg cgtgcccttg ggctccccgg
    13801 gcgcgtttgg gcggccgcat cgaattctac cgggtagggg aggcgctttt cccaaggcag
    13861 tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg
    13921 cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc
    13981 ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg
    14041 tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc
    14101 accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc
    14161 ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg
    14221 ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa
    14281 aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcaat
    14341 cgccgctagc cgccaccatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
    14401 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
    14461 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
    14521 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
    14581 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
    14641 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
    14701 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
    14761 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
    14821 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gtaagtgcta
    14881 gcccctaggc ctgaatcgaa gttcctattc ttcaaaaggt ataggaactt cgagctcgtc
    14941 gactgcagag gcctgcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga
    15001 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
    15061 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
    15121 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
    15181 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
    15241 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
    15301 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
    15361 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
    15421 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
    15481 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
    15541 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
    15601 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
    15661 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
    15721 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
    15781 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
    15841 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
    15901 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
    15961 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
    16021 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
    16081 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
    16141 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
    16201 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
    16261 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
    16321 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
    16381 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
    16441 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
    16501 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
    16561 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
    16621 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
    16681 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
    16741 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
    16801 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
    16861 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
    16921 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
    16981 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
    17041 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
    17101 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
    17161 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
