LOCUS       pAH33_-_pR26-CMVconst-Opa1       11834 bp ds-DNA     circular SYN 17-JUL-2023
DEFINITION  Allows for constitutive Cox8L-Snap expression from the murine
            ROSA26 safe harbor locus upon CRISPR/Cas9-mediated genomic
            insertion and stable selection.
ACCESSION
VERSION     .
KEYWORDS    pR26-CMVconst.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Routh E.D., Pullikuth A.K., Jin G., Chifman J., Chou J.W.,
            D'Agostino R.B., Seino K., Wada H., Print C.G., Zhang W., Lu Y. and
            Miller L.D.
  TITLE     Transcriptomic Features of T Cell-Barren Tumors Are Conserved
            Across Diverse Tumor Types
  JOURNAL   Front. Immunol., 13 February 2020
REFERENCE   2  (bases 1 to 7940)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
COMMENT     ##Imported_From-START##
            Path     :: /Users/annehakim/Downloads
            Filename :: addgene-plasmid-127373-sequence-248011.gbk
            ##Imported_From-END##
FEATURES             Location/Qualifiers
     source          1..11834
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..794
                     /note="left homology arm from intron 1 of the mouse Rosa26
                      gene (Soriano, 1999)"
                     /label="Rosa26 left arm"
     primer_bind     589..615
                     /created_by="annehakim"
                     /Sequence="CCAAAGTCGCTCTGAGTTGTTATCAGT"
                     /Tm="63.3"
                     /%GC="44.4"
                     /Hairpin_Tm="43.8"
                     /Self_Dimer_Tm="6.5"
                     /modified_by="annehakim"
                     /label="R26F1"
     primer_bind     710..731
                     /Sequence="GCCTCCTGGCTTCTGAGGACCG"
                     /Tm="66.8"
                     /%GC="68.2"
                     /Hairpin_Tm="54.5"
                     /Self_Dimer_Tm="8.9"
                     /created_by="annehakim"
                     /label="R26F2"
     misc_feature    813..916
                     /note="adenovirus major late transcript splice acceptor 
                     (Friedrich and Soriano, 1991)"
                     /label="Ad SA"
     polyA_signal    931..979
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
                     /label="HSV TK poly(A) signal"
     enhancer        1004..1305
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        1306..1508
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /label="CMV promoter"
     primer_bind     1459..1479
                     /note="Human CMV immediate early promoter, forward primer"
                     /label="CMV-F"
     primer_bind     1504..1532
                     /created_by="annehakim"
                     /Sequence="GAGCTTGGAGAATTCGAGCTCGGTACCCG"
                     /Tm="69.9"
                     /%GC="58.6"
                     /Hairpin_Tm="43.8"
                     /Self_Dimer_Tm="34.0"
                     /Color="#9911ff"
                     /modified_by="annehakim"
                     /label="1_pAH1_For_Cox8L"
     primer_bind     complement(1504..1532)
                     /created_by="annehakim"
                     /Sequence="CGGGTACCGAGCTCGAATTCTCCAAGCTC"
                     /Tm="69.9"
                     /%GC="58.6"
                     /Hairpin_Tm="83.7"
                     /Self_Dimer_Tm="36.6"
                     /Color="#7700ee"
                     /modified_by="annehakim"
                     /label="3_pAH1_Lin_pR26-CMVconst"
     gene            1518..5468
                     /created_by="annehakim"
                     /modified_by="annehakim"
                     /label="gBlock"
     primer_bind     complement(1530..1557)
                     /Sequence="CCTGCTCGCCACATGCTAGCATCCCCGG"
                     /Tm="74.1"
                     /%GC="67.9"
                     /Hairpin_Tm="58.0"
                     /Self_Dimer_Tm="27.3"
                     /created_by="annehakim"
                     /label="76_pAH33_R"
     source          <1544..>4423
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1544..4423
                     /created_by="annehakim"
                     /label="Opa1"
     misc_feature    1543^1544
                     /Original_Bases="TGA"
                     /label="TGA"
                     /note="Geneious type: Editing History Deletion"
     misc_feature    1548..4426
                     /Original_Bases="CTGTCCTGACGCCACTGCTGCTGAGGAGCCTGACCGGCTCG
                     GCCCGGCGGCTCATGGTGCCGCGGGCTCAGGTCCACTCGAAGCCGGCGCGGGAGCAGC
                     TCGGGGTCCTGGATATCACCATTGGGCTCACTTCCTGCTTCGTGTGTTGTCTTCTGCC
                     TGCGGGCTGGGTCCTGTCACACCTGGAGAGCTACAAGAAGCGGGAGTGAGGCGGATCC
                     GGCGGATCCATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGG
                     GCAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAA
                     AGGAACATCTGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGA
                     CCAGAGCCACTGATGCAGGCCACCGCCTGGCTCAACGCCTACTTTCACCAGCCTGAGG
                     CCATCGAGGAGTTCCCTGTGCCAGCCCTGCACCACCCAGTGTTCCAGCAGGAGAGCTT
                     TACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGAAGTTCGGAGAGGTCATCAGC
                     TACAGCCACCTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGTGAAAACCG
                     CCCTGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAGGGCGA
                     CCTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTGGCTGCTGGCCCAC
                     GAGGGCCACAGACTGGGCAAGCCTGGGCTGGGT"
                     /label="CTGTCCTGACGCCACTGCTGCTGAGGAGCCTGACCGGCTCGGCCCGGCGG
                     CTCATGGTGCCGCGGGCTCAGGTCCACTCGAAGCCGGCGCGGGAGCAGCTCGGGGTCC
                     TGGATATCACCATTGGGCTCACTTCCTGCTTCGTGTGTTGTCTTCTGCCTGCGGGCTG
                     GGTCCTGTCACACCTGGAGAGCTACAAGAAGCGGGAGTGAGGCGGATCCGGCGGATCC
                     ATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGGGCAAGCTGG
                     AACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAAAGGAACATC
                     TGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGACCAGAGCCA
                     CTGATGCAGGCCACCGCCTGGCTCAACGCCTACTTTCACCAGCCTGAGGCCATCGAGG
                     AGTTCCCTGTGCCAGCCCTGCACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCA
                     GGTGCTGTGGAAACTGCTGAAAGTGGTGAAGTTCGGAGAGGTCATCAGCTACAGCCAC
                     CTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCGTGAAAACCGCCCTGAGCG
                     GAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAGGGCGACCTGGACGT
                     GGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTGGCTGCTGGCCCACGAGGGCCAC
                     AGACTGGGCAAGCCTGGGCTGGGT"
                     /note="Geneious type: Editing History Replacement"
     primer_bind     4406..4437
                     /Sequence="GCTCTTCACCAGGAGAAGTGACCTGCAGGCGG"
                     /Tm="74.2"
                     /%GC="62.5"
                     /Hairpin_Tm="48.5"
                     /Self_Dimer_Tm="33.1"
                     /created_by="annehakim"
                     /label="75_pAH33_F"
     source          join(4846..>5437,<4424..4845)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     4424..4444
                     /Sequence="TGACCTGCAGGCGGATCCGCG"
                     /Tm="69.6"
                     /%GC="71.4"
                     /Hairpin_Tm="62.3"
                     /Self_Dimer_Tm="43.4"
                     /created_by="annehakim"
                     /label="7_pAH2_SDM_Cox8L_Insertion"
     intron          4537..4766
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
                     /label="chimeric intron"
     primer_bind     4749..4768
                     /note="Synthetic intron, forward primer"
                     /label="pMT2-F"
     misc_feature    4845..5412
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
                     /label="IRES"
     primer_bind     complement(5006..5023)
                     /note="IRES internal ribosome entry site, reverse primer.
                     Also called pCDH-rev"
                     /label="IRES reverse"
     primer_bind     5233..5252
                     /note="IRES internal ribosome entry site, forward primer"
                     /label="IRES-F"
     CDS             5438
                     /Color="#ff0059"
                     /created_by="annehakim"
                     /label="Insert here"
     primer_bind     complement(5452..5484)
                     /created_by="annehakim"
                     /Sequence="CATTTAAATTCATCCTACCTGCAGGGCCCGGGC"
                     /Tm="71.8"
                     /%GC="54.5"
                     /Hairpin_Tm="40.3"
                     /Self_Dimer_Tm="38.6"
                     /Color="#9911ff"
                     /modified_by="annehakim"
                     /label="2_pAH1_Rev_Cox8L"
     primer_bind     5452..5484
                     /created_by="annehakim"
                     /Sequence="GCCCGGGCCCTGCAGGTAGGATGAATTTAAATG"
                     /Tm="71.8"
                     /%GC="54.5"
                     /Hairpin_Tm="40.4"
                     /Self_Dimer_Tm="36.6"
                     /Color="#7700ee"
                     /modified_by="annehakim"
                     /label="4_pAH1_Lin_pR26-CMVconst"
     polyA_signal    5503..5551
                     /note="synthetic polyadenylation signal"
     primer_bind     6596..6615
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
                     /label="hUBCpro-F"
     CDS             6774..7373
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSR
                     LAAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVESAERAGVPAFL
                     ETSAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
                     /label="PuroR"
     primer_bind     complement(6774..6793)
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
                     /label="Puro-R"
     primer_bind     7270..7290
                     /note="Puromycin resistance gene, forward primer"
                     /label="Puro-F"
     polyA_signal    7374..7495
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(7411..7430)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     7465..7484
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     misc_feature    7552..8362
                     /note="right homology arm from intron 1 of the mouse
                     Rosa26 gene (Soriano, 1999)"
                     /label="Rosa26 right arm"
     primer_bind     complement(7639..7661)
                     /Sequence="TCTGTGGGAAGTCTTGTCCCTCC"
                     /Tm="63.5"
                     /%GC="56.5"
                     /Hairpin_Tm="52.6"
                     /Self_Dimer_Tm="10.4"
                     /created_by="annehakim"
                     /label="R26R2"
     primer_bind     complement(7926..7949)
                     /Sequence="GGAGCGGGAGAAATGGATATGAAG"
                     /Tm="61.1"
                     /%GC="50.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /created_by="annehakim"
                     /label="R26R1"
     primer_bind     complement(8436..8455)
                     /note="T7 promoter, forward primer"
                     /label="T7"
     promoter        complement(8437..8455)
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     primer_bind     complement(8462..8479)
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /label="M13 Forward"
     primer_bind     complement(8462..8478)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 fwd"
     primer_bind     complement(8471..8493)
                     /note="In lacZ gene"
                     /label="M13/pUC Forward"
     CDS             8616..8918
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
                     /label="ccdB"
     primer_bind     8797..8816
                     /note="ccdB gene, forward primer"
                     /label="ccdB-fwd"
     CDS             9267..10061
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                      (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label="NeoR/KanR"
     primer_bind     complement(9321..9340)
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     primer_bind     9931..9950
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     regulatory      10261..10270
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation
                      of translation (Kozak, 1987)"
     CDS             10267..10641
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
                     DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPW
                     GREFALRDPAGNCVHFVAEEQD"
                     /label="BleoR"
     rep_origin      10779..11367
                     /direction="RIGHT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     11268..11287
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     primer_bind     11521..11538
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     protein_bind    11655..11676
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     promoter        11691..11721
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    11729..11745
                     /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)."
                     /label="lac operator"
     primer_bind     11734..11756
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     primer_bind     11753..11769
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     11753..11769
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     promoter        11787..11805
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /label="SP6 promoter"
     primer_bind     11787..11804
                     /note="SP6 promoter, forward primer"
                     /label="SP6"
ORIGIN      
        1 cagttaacgg cagccggagt gcgcagccgc cggcagcctc gctctgccca ctgggtgggg
       61 cgggaggtag gtggggtgag gcgagctgga cgtgcgggcg cggtcggcct ctggcggggc
      121 gggggagggg agggagggtc agcgaaagta gctcgcgcgc gagcggccgc ccaccctccc
      181 cttcctctgg gggagtcgtt ttacccgccg ccggccgggc ctcgtcgtct gattggctct
      241 cggggcccag aaaactggcc cttgccattg gctcgtgttc gtgcaagttg agtccatccg
      301 ccggccagcg ggggcggcga ggaggcgctc ccaggttccg gccctcccct cggccccgcg
      361 ccgcagagtc tggccgcgcg cccctgcgca acgtggcagg aagcgcgcgc tgggggcggg
      421 gacgggcagt agggctgagc ggctgcgggg cgggtgcaag cacgtttccg acttgagttg
      481 cctcaagagg ggcgtgctga gccagacctc catcgcgcac tccggggagt ggagggaagg
      541 agcgagggct cagttgggct gttttggagg caggaagcac ttgctctccc aaagtcgctc
      601 tgagttgtta tcagtaaggg agctgcagtg gagtaggcgg ggagaaggcc gcacccttct
      661 ccggaggggg gaggggagtg ttgcaatacc tttctgggag ttctctgctg cctcctggct
      721 tctgaggacc gccctgggcc tgggagaatc ccttccccct cttccctcgt gatctgcaac
      781 tccagtcttt ctagaatcga tctcgagaac tgtagggcgc agtagtccag ggtttccttg
      841 atgatgtcat acttatcctg tccctttttt ttccacagct cgcggttgag gacaaactct
      901 tcgcggtctt tccagtggtt aattagggat cggcaataaa aagacagaat aaaacgcacg
      961 ggtgttgggt cgtttgttca tcgatgccct ttcgtcttca ttccgttaca taacttacgg
     1021 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1081 atgttcccat agtaacgcca atagggactt tccattgacg tcaatggtgg agtatttacg
     1141 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     1201 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     1261 tcctacttgg cagtacatct actattagtc atcgctatta ccatggtgat gcggttttgg
     1321 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccaccca
     1381 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1441 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1501 gcagagcttg gagaattcga gctcggtacc cggggatgct agcatgtggc gagcaggtcg
     1561 ggcggccgtg gcctgtgaag tctgccaatc cttagtgaaa cacagctctg gaatacaaag
     1621 aaacgtaccg ctccaaaaac tccatctggt ttcacgaagt atttatcgtt cacatcatcc
     1681 tgccctcaag cttcaaagac cccaactaag gacaccattt cagcagttct cttctctaac
     1741 tcacctttca ttacataaat tgaaactttc tccaattaaa tatggctacc agccccgcag
     1801 gaacttttgg ccagcaaggt tagctgcaag actcttaaaa cttcgatata tcatactggg
     1861 atctgctgtt ggaggtggct atacagccaa aaaaaccttc gatgaatgga aagatatgat
     1921 accagacctt agtgactata agtggattgt gcctgacttt atatgggaaa ttgatgagta
     1981 tattgatttg gagaaaatta gaaaagccct tcccagctca gaagaccttg ccagtttagc
     2041 tcccgacttg gacaagatta ctgagagcct cagcttgttg aaggacttct tcactgcagg
     2101 ttcacctgga gaaacagcat ttcgagcaac agatcatgga tctgaaagtg acaagcatta
     2161 caggaaggtg tcagacaaag aaaagattga ccaacttcaa gaagaacttc tgcatactca
     2221 gttaaagtat cagaggatct tggagcgcct ggaaaaggag aacaaagagc tgaggaagct
     2281 ggtgctgcag aaggacgaca aaggcatcca ccacaggaag ctcaagaaat ctttgattga
     2341 tatgtattct gaagttcttg atgttctttc tgattatgat gccagttaca atacacaaga
     2401 tcacctacca cgggttgttg tggttggaga tcagagtgct gggaaaacca gtgtgctgga
     2461 aatgattgct caggcccgga tcttcccgag agggtccggc gagatgatga cacgctctcc
     2521 agtgaaggtg actctcagtg aaggccctca ccatgtggcc ttgtttaaag atagctctcg
     2581 ggaatttgat ctcaccaagg aggaagatct tgcagcatta agacatgaaa tcgaactccg
     2641 aatgaggaaa aatgtgaaag aaggttgtac tgttagcccc gagaccatat ctctaaatgt
     2701 caaaggccct gggctgcaga ggatggtgct cgtggacttg cctggtgtca tcaacactgt
     2761 gacatcaggc atggctcccg acacaaagga aactattttc agtatcagca aagcttacat
     2821 gcagaatcct aacgccatca tcctgtgcat ccaagacgga tccgtagatg ctgagcgcag
     2881 tattgttaca gacttggtca gtcaaatgga tcctcatgga agaagaacca tatttgtttt
     2941 gaccaaagta gacctggcag aaaaaaatgt agccagtcca agcaggatac aacagataat
     3001 tgaaggcaag ctcttcccaa tgaaagctct gggttatttt gctgtcgtaa caggaaaagg
     3061 aaacagctct gaaagtattg aagctataag agagtatgaa gaggaatttt ttcagaattc
     3121 aaaactgcta aagacaagca tgctaaaggc acaccaggtc accacgagaa atctcagcct
     3181 tgctgtgtca gactgctttt ggaaaatggt tcgagagtca gttgaacaac aggctgatag
     3241 ttttaaagcc acgcgcttta acctagagac ggaatggaag aataactacc cgcgcctgcg
     3301 agagctcgac aggaatgaac tctttgaaaa agctaaaaat gagatcctcg atgaggtcat
     3361 cagtctgagc caggtcacgc caaagcactg ggaggaaatc ctgcagcaat ccctgtggga
     3421 acgagtgtca acacatgtga ttgagaacat ctaccttcca gctgcccaga ccatgaattc
     3481 gggaacattt aacaccacag tagacatcaa gcttaaacag tggactgaca agcagcttcc
     3541 taataaagca gtcgaggttg cctgggagac tctacaagag gaattttccc gcttcatgac
     3601 agaacccaaa ggaaaggaac acgacgacat atttgacaaa cttaaggagg ctgtgaagga
     3661 ggagagtatc aagcggcaca agtggaacga ctttgccgag gatagcttga gggttattca
     3721 gcacaatgct ttggaagacc ggtccatatc agataagcaa cagtgggatg cagccattta
     3781 cttcatggaa gaggcgcttc aaggtcgtct caaggatact gaaaatgcta ttgaaaacat
     3841 gattgggcca gactggaaaa agaggtggat gtactggaag aatcggaccc aagagcagtg
     3901 tgttcacaac gaaaccaaga acgagttgga gaagatgctg aaggttaatg atgagcaccc
     3961 agcttacctg gcaagtgatg agattaccac agtccggaag aacctggagt ctcgaggagt
     4021 ggaagtcgat ccaagcttga ttaaggatac ttggcatcaa gtttatagaa gacatttctt
     4081 aaaaacagct ctaaatcatt gtaacctttg tcgcagaggt ttttattact accagaggca
     4141 ttttatagat tctgagctgg aatgcaatga cgtggtcctg ttttggcgaa tacagcgcat
     4201 gctcgctatc actgccaata cattaaggca gcagcttaca aacactgaag ttaggcgact
     4261 agagaaaaac gttaaagagg tattagaaga ttttgcagaa gacggtgaga agaaggttaa
     4321 attgctcact ggcaaacgag ttcagctggc agaagatctc aagaaagtta gagaaattca
     4381 agaaaagctt gatgctttca ttgaagctct tcaccaggag aagtgacctg caggcggatc
     4441 cgcgtttaaa ctcgaggtta attaatgagc ggccgcatag ataactgatc cagtgtgctg
     4501 gaattaattc gctgtctgcg agggccagct gttggggtga gtactccctc tcaaaagcgg
     4561 gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct
     4621 ggcccgcggt gatgcctttg agggtggccg cgtccatctg gtcagaaaag acaatctttt
     4681 tgttgtcaag cttgaggtgt ggcaggcttg agatctggcc atacacttga gtgacaatga
     4741 catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc aggtcgagca
     4801 tgcatctagg gcggccaatt ccgcccctct cccccccccc cttttccctc ccccccccct
     4861 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
     4921 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     4981 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     5041 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
     5101 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     5161 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     5221 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
     5281 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
     5341 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     5401 acacgatgat aagcttgcca caacccggga taattcctct aggttaatta agcccgggcc
     5461 ctgcaggtag gatgaattta aatgtttaaa cacgcgttag gaaataaaag atctttattt
     5521 tcattagatc tgtgtgttgg ttttttgtgt gggcgcgccg ggttttggcg cctcccgcgg
     5581 gcgcccccct cctcacggcg agcgctgcca cgtcagacga agggcgcagc gagcgtcctg
     5641 atccttccgc ccggacgctc aggacagcgg cccgctgctc ataagactcg gccttagaac
     5701 cccagtatca gcagaaggac attttaggac gggacttggg tgactctagg gcactggttt
     5761 tctttccaga gagcggaaca ggcgaggaaa agtagtccct tctcggcgat tctgcggagg
     5821 gatctccgtg gggcggtgaa cgccgatgat tatataagga cgcgccgggt gtggcacagc
     5881 tagttccgtc gcagccggga tttgggtcgc ggttcttgtt tgtggatcgc tgtgatcgtc
     5941 acttggtgag tagcgggctg ctgggctggc cggggctttc gtggccgccg ggccgctcgg
     6001 tgggacggaa gcgtgtggag agaccgccaa gggctgtagt ctgggtccgc gagcaaggtt
     6061 gccctgaact gggggttggg gggagcgcag caaaatggcg gctgttcccg agtcttgaat
     6121 ggaagacgct tgtgaggcgg gctgtgaggt cgttgaaaca aggtgggggg catggtgggc
     6181 ggcaagaacc caaggtcttg aggccttcgc taatgcggga aagctcttat tcgggtgaga
     6241 tgggctgggg caccatctgg ggaccctgac gtgaagtttg tcactgactg gagaactcgg
     6301 tttgtcgtct gttgcggggg cggcagttat ggcggtgccg ttgggcagtg cacccgtacc
     6361 tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg ttctgttggc ttataatgca
     6421 gggtggggcc acctgccggt aggtgtgcgg taggcttttc tccgtcgcag gacgcagggt
     6481 tcgggcctag ggtaggctct cctgaatcga caggcgccgg acctctggtg aggggaggga
     6541 taagtgaggc gtcagtttct ttggtcggtt ttatgtacct atcttcttaa gtagctgaag
     6601 ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt tttgtgaagt tttttaggca
     6661 ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc agtgttagac tagtaaattg
     6721 tccgctaaat tctggccgtt tttggctttt ttgttagacc aagtttggcc accatgaccg
     6781 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     6841 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     6901 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     6961 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     7021 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     7081 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     7141 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     7201 tcgtgctccc cggagtggag tcggccgagc gcgccggggt gcccgccttc ctggagacct
     7261 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     7321 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaaacttgt
     7381 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     7441 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttagggta
     7501 ccgagctcgg atccactagt aacggccgcc agtgtgctgg aattcgccct tagatgggcg
     7561 ggagtcttct gggcaggctt aaaggctaac ctggtgtgtg ggcgttgtcc tgcaggggaa
     7621 ttgaacaggt gtaaaattgg agggacaaga cttcccacag attttcggtt ttgtcgggaa
     7681 gttttttaat aggggcaaat aaggaaaatg ggaggatagg tagtcatctg gggttttatg
     7741 cagcaaaact acaggttatt attgcttgtg atccgcctcg gagtattttc catcgaggta
     7801 gattaaagac atgctcaccc gagttttata ctctcctgct tgagatcctt actacagtat
     7861 gaaattacag tgtcgcgagt tagactatgt aagcagaatt ttaatcattt ttaaagagcc
     7921 cagtacttca tatccatttc tcccgctcct tctgcagcct tatcaaaagg tattttagaa
     7981 cactcatttt agccccattt tcatttatta tactggctta tccaacccct agacagagca
     8041 ttggcatttt ccctttcctg atcttagaag tctgatgact catgaaacca gacagattag
     8101 ttacatacac cacaaatcga ggctgtagct ggggcctcaa cactgcagtt cttttataac
     8161 tccttagtac actttttgtt gatcctttgc cttgatcctt aattttcagt gtctatcacc
     8221 tctcccgtca ggtggtgttc cacatttggg cctattctca gtccagggag ttttacaaca
     8281 atagatgtat tgagaatcca acctaaagct taactttcca ctcccatgaa tgcctctctc
     8341 ctttttctcc atttataaac tgaagggcga attctgcaga tatccatcac actggcggcc
     8401 gctcgatcga gcatgcatct agagggccca attcgcccta tagtgagtcg tattacaatt
     8461 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     8521 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     8581 gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac acctataaaa
     8641 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgccggggc
     8701 gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     8761 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     8821 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     8881 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg agattatcaa
     8941 aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa cggtgctgac
     9001 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     9061 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     9121 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     9181 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agctctgatc
     9241 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     9301 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     9361 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     9421 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     9481 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     9541 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     9601 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     9661 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     9721 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     9781 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     9841 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     9901 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     9961 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
    10021 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt
    10081 tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc
    10141 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
    10201 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgaggagg
    10261 gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
    10321 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
    10381 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
    10441 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
    10501 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
    10561 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
    10621 gtggccgagg agcaggactg acacgtgcta aaacttcatt tttaatttaa aaggatctag
    10681 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
    10741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
    10801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
    10861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
    10921 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
    10981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
    11041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
    11101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
    11161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
    11221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
    11281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
    11341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
    11401 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
    11461 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
    11521 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
    11581 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
    11641 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
    11701 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
    11761 agctatgacc atgattacgc caagctattt aggtgacact atagaatact caagctatgc
    11821 atcaagcttg gtac
//
