LOCUS       pFSynW_syt1_C2AB       10168 bp DNA     circular SYN 25-AUG-2023
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10168)
  AUTHORS   nmehta22
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2023 from SnapGene 7.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..10168
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /note="color: #ffe4c4"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
                     /note="color: #ffe4c4"
     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."
                     /note="color: #ffe4c4"
     misc_feature    2440..2557
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
                     /note="color: #ffe4c4"
     primer_bind     2582..2624
                     /label=NC32 For IF
                     /note="color: black; sequence: 
                     GATCCAGTTTGGTTAATTAAagtgcaagtggttttaggaccag"
     promoter        2602..3048
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     3033..3092
                     /label=80KF3
                     /note="color: black; sequence: 
                     gtgcctgagagcgcagGTACGGCTAGCGCCACCATGAACCAATGTTTGTTCCAATCGAC
                     C; added: 2023-06-30"
     primer_bind     3042..3090
                     /label=Fragment.FOR
                     /note="color: black; sequence: 
                     agcgcagGTACGGCTAGCGCCACCATGAACCAATGTTTGTTCCAATCGA; added: 
                     2023-06-21"
     misc_feature    3060..3065
                     /label=KOZAK
                     /note="color: #a6acb3"
     CDS             3066..4832
                     /codon_start=1
                     /label=syt1 C2AB 80K-421-msGFP
                     /note="color: #ff9900"
                     /translation="MNQCLFQSTNNTQGSENGGNNAINMQDVNDLGSTMTDQALNDDDA
                     ETGLTDGEENEEPTEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVK
                     VFLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIG
                     EFKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLK
                     KMDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVV
                     TVLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLA
                     VKKGSGSSGSSGSSGSSMDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFK
                     DDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNG
                     IKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMV
                     LLEFVTAAGITHG"
     CDS             3066..3068
                     /codon_start=1
                     /label=rSyt1_FL
                     /note="color: #993366"
                     /translation="M"
     CDS             3069..3257
                     /codon_start=1
                     /label=syt1 80-421-Juxta Lys
                     /note="color: #a6acb3"
                     /translation="NQCLFQSTNNTQGSENGGNNAINMQDVNDLGSTMTDQALNDDDAE
                     TGLTDGEENEEPTEEEKL"
     CDS             3258..4094
                     /codon_start=1
                     /label=rSyt1_FL
                     /note="color: #993366"
                     /translation="GKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKVFLLP
                     DKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGEFKVP
                     MNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKKMDVG
                     GLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVTVLDY
                     DKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLAVKK"
     misc_feature    3258..4094
                     /label=Syt1 C2AB 80-421
                     /note="color: #a6acb3"
     primer_bind     3485..3508
                     /label=Primer 1
                     /note="color: black; sequence: attaggtggcaaaaccctggtgat; 
                     added: 2020-09-16"
     primer_bind     complement(3485..3508)
                     /label=Primer 2
                     /note="color: black; sequence: atcaccagggttttgccacctaat; 
                     added: 2020-09-16"
     primer_bind     4074..4159
                     /label=msGFP-FLAG 60 F (EC13459)
                     /note="color: black; sequence: 
                     GCCATGCTGGCTGTCAAGAAGGGATCCGGAAGTTCGGGGTCCAGCGGTTCTTCAGGCAG
                     TTCCATGGATAGCACTGAGAGCCTGTT; added: 2020-09-21"
     primer_bind     complement(4075..4094)
                     /label=Syt1 C2AB 80 R
                     /note="color: black; sequence: 
                     ATAAATACCTAAGCTTttacttcttgacagccagcatgg; added: 2020-09-04"
     CDS             4095..4136
                     /label=GS Linker
                     /note="color: #993366; direction: RIGHT"
     misc_feature    4095..4100
                     /label=linker for pre-prolactin
                     /note="found in original sequence"
                     /note="color: #a6acb3"
     primer_bind     4135..4172
                     /label=DSTES
                     /note="Use together with BL14 to amplify msGFP, msYFP, and 
                     msCFP with the improved N-terminal codons from A24. 
                     Subclone the PCR product back into the same vector using 
                     BamHI and NotI."
                     /note="color: black; sequence: 
                     CCGGGATCCACCGGTCGCCACCATGGATAGCACTGAGAGCCTGTTCACCGGGGTGGTG"
     CDS             4137..4832
                     /codon_start=1
                     /label=msGFP
                     /note="color: #05fd14"
                     /translation="MDSTESLFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTITFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHG"
     CDS             4137..4154
                     /codon_start=1
                     /product="""An N-terminal MDSTES peptide in place of 
                     MVSKGEE """"In particular, the widely used red FP mCherry 
                     contains the N- and C-terminal peptides of EGFP (12), and 
                     we find that the N-terminal peptide of EGFP can promote 
                     cytotoxicity of mCherry during bacterial expression.""
                       A photostable monomeric superfolder GFPFernando Valbuena,
                     Ivy Fizgerald, Rita L. Strack, Neal Andruska, Luke Smith, 
                     Benjamin S. Glick 
                     "
                     /label=N-terminus mutation
                     /note="color: #993366"
                     /translation="MDSTES"
     primer_bind     4140..4163
                     /label=new N GFP lin F
                     /note="color: black; sequence: 
                     GCCATTTGCTAAACCTGTTAGAGCTGATAGCACTGAGAGCCTGTTCACC"
     CDS             4224..4226
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (1/8) S30R
                     /note="color: #993366"
                     /translation="R"
     CDS             4251..4253
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (2/8) Y39N
                     /note="color: #993366"
                     /translation="N"
     primer_bind     complement(4264..4286)
                     /label=msGFP N term R
                     /note="color: black; sequence: GGTGGTGCAGATGAACTTCAGGG; 
                     added: 2020-08-31"
     CDS             4431..4433
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (3/8) F99T
                     /note="color: #993366"
                     /translation="T"
     CDS             4449..4451
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (4/8) N105T
                     /note="color: #993366"
                     /translation="T"
     CDS             4569..4571
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850. 
                     Reversion of this mutation (F145Y) restores photostability 
                     of superfolding mutant with only a minor effect on folding 
                     Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS.
                     Engineering and characterization of a superfolder green 
                     fluorescent protein. Nat Biotechnol 2006;24:79–88.
                     "
                     /label=superfolder mutation (5/8) Y145F
                     /note="color: #993366"
                     /translation="F"
     CDS             4593..4595
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (6/8) M153T
                     /note="color: #993366"
                     /translation="T"
     primer_bind     complement(4616..4640)
                     /label=BQ13
                     /note="color: black; sequence: GCGGATCTTGAAGTTGGCCTTGATG"
     CDS             4623..4625
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850"
                     /label=superfolder mutation (7/8) V163A
                     /note="color: #993366"
                     /translation="A"
     CDS             4647..4649
                     /codon_start=1
                     /product="Shagin DA, Barsova EV, Yanushevich YG, Fradkov 
                     AF, Lukyanov KA, Labas YA, Semenova TN, Ugalde JA, Meyers 
                     A, Nunez JM, Widder EA, Lukyanov SA, Matz MV. GFP-like 
                     proteins as ubiquitous metazoan superfamily: evolution of 
                     functional features and structural complexity. Mol Biol 
                     Evol 2004;21(5):841–850."
                     /label=superfolder mutation (8/8) I171V
                     /note="color: #993366"
                     /translation="V"
     primer_bind     4707..4722
                     /label=msGFP C term F
                     /note="color: black; sequence: GGCCCCGTGCTGCTGC; added: 
                     2020-08-31"
     CDS             4752..4754
                     /codon_start=1
                     /product="Costantini LM, Fossati M, Francolini M, Snapp EL.
                     Assessing the tendency of fluorescent proteins to 
                     oligomerize under physiologic conditions. Traffic 
                     2012;13(5):643–649.
                     Zacharias DA, Violin JD, Newton AC, Tsien RY. Partitioning 
                     of lipid-modified monomeric GFPs into membrane microdomains
                     of live cells. Science 2002;296:913–916.
                     "
                     /label=monomerizing mutation A206K
                     /note="color: #993366"
                     /translation="K"
     primer_bind     complement(4815..4832)
                     /label=BL14
                     /note="This oligo (used with BL13) will replace the linker 
                     MDELYK to GSSGSS in monomeric superfolder GFP, CFP and 
                     YFP."
                     /note="color: black; sequence: 
                     GATCTAGAGTCGCGGCCGCTTTAcccgctggagccggagctGCCGTGAGTGATCCCGGC
                     "
     primer_bind     complement(4817..4853)
                     /label=Fragment.REV
                     /note="color: black; sequence: 
                     AAGCTTGATATCGAATTCTTaGCCGTGAGTGATCCCG; added: 2023-06-21"
     primer_bind     complement(4822..4832)
                     /label=BL24
                     /note="This oligo is used to amplify msfFPs (Green, Cyan, 
                     Yellow) from the pMSGFP vector into the NT-HT vector for 
                     the Tub1 fussion.  The cut sites are BspEI for the 
                     N-terminal and SacI for the C-terminal. Use this oligo with
                     BL23."
                     /note="color: black; sequence: 
                     GGTTCCAGAGGGAGCACCGGAGCTCGTcccgctggagccggagctGCCGTGAGTGA"
     CDS             4827..4829
                     /codon_start=1
                     /product="Tsien RY. The green fluorescent protein. Annu Rev
                     Biochem 1998;67:509–544."
                     /label=monomeric mutation L231H
                     /note="color: #993366"
                     /translation="H"
     misc_feature    4860..5448
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
                     /note="color: #ffffff"
     CDS             complement(5331..5342)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /note="color: #cc99b2
                     Cleavage site after base 5331"
                     /translation="IEGR"
     primer_bind     complement(5451..5467)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     LTR             5973..6153
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /note="color: #ffe4c4"
     polyA_signal    6185..6409
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      6455..6883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        6897..7226
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      7077..7212
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     promoter        7274..7321
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
                     /note="color: #ffffff; direction: RIGHT"
     CDS             7340..7714
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /note="color: #ccffcc"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7844..7965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(8014..8030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    8038..8054
                     /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)."
                     /note="color: #31849b"
     promoter        complement(8062..8092)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 8062 .. 8068 / #ffffff / -10
                        2: 8069 .. 8086 / #ffffff
                        3: 8087 .. 8092 / #ffffff / -35"
     protein_bind    8107..8128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(8416..9004)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(9175..10035)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1:  9175 ..  9966 / #ccffcc
                        2:  9967 .. 10035 / #ccffcc / signal sequence
                     Cleavage site after base 9967"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(10036..10140)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
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 aagtgcaagt ggttttagga ccaggatgag gcggggtggg
     2641 ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa
     2701 attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca
     2761 ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc
     2821 cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgtaaac tccccttccc
     2881 ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc
     2941 gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct
     3001 cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcaggt acggctagcg
     3061 ccaccatgaa ccaatgtttg ttccaatcga ccaacaacac tcaggggtcg gaaaacggag
     3121 gaaacaacgc cattaacatg caagacgtga acgacttagg gtcgaccatg actgatcagg
     3181 cccttaatga tgacgatgct gaaaccggac tgactgatgg agaagaaaac gaagagccca
     3241 ctgaagagga gaaactggga aagctccaat attcactgga ctatgacttc cagaataacc
     3301 agctgttggt gggaatcatc caggctgctg aactgcccgc cctggacatg gggggtacat
     3361 ccgatccata cgtcaaagtc ttcctgctgc ctgacaaaaa gaagaaattt gagactaaag
     3421 tccaccggaa aaccctcaat ccagtcttca atgaacaatt tactttcaag gtaccctact
     3481 cggaattagg tggcaaaacc ctggtgatgg ctgtgtatga ctttgatcgc ttctccaagc
     3541 acgacatcat cggagagttc aaagttccta tgaacaccgt ggattttggc catgtgaccg
     3601 aggagtggcg cgatctccag agcgctgaga aagaagagca agagaaactg ggtgacatct
     3661 gcttctccct ccgctacgtc cctactgccg gcaaactgac tgttgtcatt ctggaagcca
     3721 agaacctgaa gaagatggat gtgggtggct tatctgatcc ctacgtgaag attcacctga
     3781 tgcagaacgg taagaggctg aagaagaaaa agacgacgat taagaagaac acactcaacc
     3841 cctactacaa cgagtccttc agctttgaag ttccgttcga gcaaatccag aaagtgcaag
     3901 tggtggtaac tgttttggac tatgacaaga ttggcaagaa cgacgccatc ggaaaagtct
     3961 tcgttggtta caacagcact ggggcggagc tgcgacactg gtcagacatg ctggccaacc
     4021 cccggcgacc catcgcacag tggcacactc tgcaggtaga ggaggaggtt gatgccatgc
     4081 tggctgtcaa gaagggatcc ggaagttcgg ggtccagcgg ttcttcaggc agttccatgg
     4141 atagcactga gagcctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     4201 taaacggcca caagttcagc gtgcgcggcg agggcgaggg cgatgccacc aacggcaagc
     4261 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     4321 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     4381 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc accttcaagg
     4441 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4501 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4561 agtacaactt caacagccac aacgtctata tcaccgccga caagcagaag aacggcatca
     4621 aggccaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc gccgaccact
     4681 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     4741 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     4801 agttcgtgac cgccgccggg atcactcacg gctaagaatt cgatatcaag cttatcgata
     4861 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4921 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     4981 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     5041 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     5101 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     5161 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5221 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     5281 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5341 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5401 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     5461 gacctcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg
     5521 ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc
     5581 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     5641 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt
     5701 ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga
     5761 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg
     5821 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
     5881 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
     5941 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
     6001 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     6061 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     6121 gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg ctgatcagcc
     6181 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     6241 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     6301 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     6361 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     6421 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     6481 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6541 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6601 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6661 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6721 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6781 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6841 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     6901 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     6961 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     7021 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     7081 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     7141 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     7201 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     7261 ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     7321 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     7381 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     7441 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     7501 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     7561 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     7621 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     7681 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt tcgattccac
     7741 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     7801 cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc
     7861 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     7921 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc
     7981 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     8041 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     8101 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     8161 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     8221 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     8281 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     8341 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     8401 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     8461 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     8521 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     8581 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     8641 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     8701 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     8761 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     8821 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     8881 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     8941 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     9001 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     9061 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     9121 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     9181 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     9241 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     9301 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     9361 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     9421 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     9481 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     9541 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     9601 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     9661 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     9721 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     9781 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     9841 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     9901 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     9961 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
    10021 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
    10081 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
    10141 cacatttccc cgaaaagtgc cacctgac
//
