LOCUS       sequence-384274-       10532 bp    DNA     circular SYN 14-APR-2025
DEFINITION  CAR featuring: GMCSF (sig. peptide); CD8A & CD8A (hinge & TM); 4-1BB
            & CD3-zeta (signaling domains); anti-CD19 from FMC63 in VL-VH order 
            and 218 linker (scFv). GFP-Zeo for selection and monitoring..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10532)
  AUTHORS   Silva G, Rodrigues AF, Ferreira S, Matos C, Eleuterio RP, Marques G,
            Kucheryava K, Lemos AR, Sousa PMF, Castro R, Barbas A, Simao D, 
            Alves PM
  TITLE     Novel scFv against Notch Ligand JAG1 Suitable for Development of 
            Cell Therapies toward JAG1-Positive Tumors.
  JOURNAL   Biomolecules. 2023 Mar 2;13(3):459. doi: 10.3390/biom13030459.
  PUBMED    36979394
REFERENCE   2  (bases 1 to 10532)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Biomolecules."; date: "2023-03-2"; volume: "13(3):459. doi"; pages:
            " 10.3390/biom13030459"
COMMENT     Sequence Label: pRRL.SIN.EF1A.CD19-FMC63.218.CAR-2G
FEATURES             Location/Qualifiers
     source          1..10532
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1802..1919
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1944..3122
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2175..3113
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3070..3090
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        4649..4667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    4702..5267
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4717..5269
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4861..4878)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5088..5107
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(5275..5277,5278..5280,5281..5991)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5275..5277,5278..5280,5281..5991)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5320..5341)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5581..5600)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5928..5949
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5995..6369
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    6376..6964
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6429..6449)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6847..6858)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             7051..7284
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    7356..7477
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7393..7412)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7447..7466
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      7517..7652
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(7673..7691)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7701..7718)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7701..7717)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7710..7732)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7859..8314
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7946..7965)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8156..8177
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8340..8444
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8445..8513,8514..9305)
                     /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     complement(8663..8682)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9476..10064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9965..9984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10218..10235
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    10352..10373
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(10388..10393,10394..10411,10412..10418)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    10426..10442
                     /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)."
     primer_bind     10431..10453
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     10450..10466
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     10450..10466
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     10485..10505
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        10487..10505
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg cctctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
     1741 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcggttaa
     1801 cttttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     1861 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta
     1921 tcgatcacga gactagcctc gagggctccg gtgcccgtca gtgggcagag cgcacatcgc
     1981 ccacagtccc cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt
     2041 ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg
     2101 ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg
     2161 ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt
     2221 atggcccttg cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg
     2281 agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc
     2341 ctcgtgcttg agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc
     2401 accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac
     2461 ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca
     2521 ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat
     2581 gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag
     2641 ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg
     2701 caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg
     2761 ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca
     2821 cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc
     2881 gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt
     2941 ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta
     3001 ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt
     3061 ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt
     3121 gaggaattag cttggtacga attcgccacc atgcttctcc tggtgacaag ccttctgctc
     3181 tgtgagttac cacacccagc attcctcctg atcccagaca tccagatgac acagactaca
     3241 tcctccctgt ctgcctctct gggagacaga gtcaccatca gttgcagggc aagtcaggac
     3301 attagtaaat atttaaattg gtatcagcag aaaccagatg gaactgttaa actcctgatc
     3361 taccatacat caagattaca ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga
     3421 acagattatt ctctcaccat tagcaacctg gagcaagaag atattgccac ttacttttgc
     3481 caacagggta atacgcttcc gtacacgttc ggagggggga ctaagttgga aataacaggc
     3541 tccacctctg gatccggcaa gcccggatct ggcgagggat ccaccaaggg cgaggtgaaa
     3601 ctgcaggagt caggacctgg cctggtggcg ccctcacaga gcctgtccgt cacatgcact
     3661 gtctcagggg tctcattacc cgactatggt gtaagctgga ttcgccagcc tccacgaaag
     3721 ggtctggagt ggctgggagt aatatggggt agtgaaacca catactataa ttcagctctc
     3781 aaatccagac tgaccatcat caaggacaac tccaagagcc aagttttctt aaaaatgaac
     3841 agtctgcaaa ctgatgacac agccatttac tactgtgcca aacattatta ctacggtggt
     3901 agctatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agcggccgca
     3961 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg
     4021 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
     4081 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc
     4141 ctgtcactgg ttatcaccct ttactgcaaa cggggcagaa agaaactcct gtatatattc
     4201 aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga
     4261 tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac
     4321 gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga
     4381 gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg
     4441 agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag
     4501 gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt
     4561 taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg
     4621 ccccctcgct aacgtacgcc cgcgaaatta atacgactca ctatagggag accacaacgg
     4681 tttccctcta gcgggatcaa ttccgccccc cccctaacgt tactggccga agccgcttgg
     4741 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     4801 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     4861 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     4921 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     4981 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     5041 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     5101 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     5161 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     5221 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatac tagtatggtg
     5281 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     5341 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     5401 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     5461 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     5521 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     5581 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     5641 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     5701 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     5761 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     5821 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     5881 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     5941 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggcatggcc
     6001 aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc
     6061 tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc
     6121 cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc
     6181 ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg
     6241 tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg
     6301 gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag
     6361 caggactgag tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     6421 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     6481 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     6541 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     6601 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     6661 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     6721 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc
     6781 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     6841 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     6901 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     6961 ccgcctgtaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     7021 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     7081 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     7141 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     7201 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     7261 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     7321 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     7381 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     7441 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     7501 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     7561 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     7621 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     7681 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     7741 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     7801 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     7861 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     7921 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     7981 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     8041 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     8101 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     8161 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     8221 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     8281 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     8341 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     8401 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     8461 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     8521 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     8581 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     8641 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     8701 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     8761 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     8821 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     8881 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     8941 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     9001 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     9061 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     9121 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     9181 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     9241 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     9301 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     9361 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     9421 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     9481 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     9541 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     9601 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     9661 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     9721 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     9781 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     9841 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     9901 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     9961 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
    10021 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
    10081 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
    10141 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
    10201 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
    10261 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
    10321 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
    10381 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
    10441 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
    10501 aaagggaaca aaagctggag ctgcaagctt aa
//
