LOCUS       sequence-267565-        9631 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral GFP Reporter assay for determining appropriate dose of 
            puromycin, blasticidin, and hygromycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9631)
  AUTHORS   DeWeirdt PC, Sanson KR, Sangree AK, Hegde M, Hanna RE, Feeley MN, 
            Griffith AL, Teng T, Borys SM, Strand C, Joung JK, Kleinstiver BP, 
            Pan X, Huang A, Doench JG
  TITLE     Optimization of AsCas12a for combinatorial genetic screens in human 
            cells.
  JOURNAL   Nat Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6.
  PUBMED    32661438
REFERENCE   2  (bases 1 to 9631)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 13. pii: 10.1038/s41587-020-0600-6. doi: 
            10.1038/s41587-020-0600-6."
COMMENT     Sequence Label: pRosetta_v2
FEATURES             Location/Qualifiers
     source          1..9631
                     /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    1801..1918
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    1936..1956
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1960..2460
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2379..2397
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    2461..2485
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2492..3514
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2492..3514
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3524..3577
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3578..3973
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             3983..4039
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4040..4636
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4040..4059)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4536..4556
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4646..4711
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(4712..4714,4715..4717,4718..5431)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4712..4714,4715..4717,4718..5428)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4757..4778)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5018..5037)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5365..5386
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5432..5456)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    5474..6062
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5527..5547)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5945..5956)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6150..6383
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6455..6576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6492..6511)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6546..6565
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6616..6751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6771..6790)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6772..6790)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6800..6817)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6800..6816)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6809..6831)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6958..7413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7045..7064)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7255..7276
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7439..7543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7544..7612,7613..8404)
                     /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(7762..7781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8575..9163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9064..9083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9317..9334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9451..9472
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9487..9492,9493..9510,9511..9517)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9525..9541
                     /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     9530..9552
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9549..9565
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9549..9565
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9584..9604
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9586..9604
                     /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 ccgctgatct 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 tatcgctagc
     1801 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     1861 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttac
     1921 tagtgattat cggatcaact ttgtatagaa aagttggggt tgcgcctttt ccaaggcagc
     1981 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc
     2041 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct
     2101 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg
     2161 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca
     2221 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc
     2281 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg
     2341 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct
     2401 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag
     2461 gcaagtttgt acaaaaaagc aggctgccac catgaaaaag cctgaactca ccgcgacgtc
     2521 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     2581 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     2641 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     2701 cgcgctcccg attccggaag tgcttgacat tggggaattt agcgagagcc tgacctattg
     2761 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     2821 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     2881 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     2941 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3001 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3061 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3121 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3181 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3241 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3301 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     3361 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     3421 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     3481 ccgacgcccc agcactcgtc cgagggcaaa ggaaggaagc ggagagggca ggggaagtct
     3541 tctaacatgc ggggacgtgg aggaaaatcc cggccccatg gccaagcctt tgtctcaaga
     3601 agaatccacc ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga
     3661 ctacagcgtc gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt
     3721 atatcatttt actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc
     3781 ggcagctggc aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag
     3841 cccctgcgga cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt
     3901 gaaggacagt gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta
     3961 tgtgtgggag ggcggaagcg gagccacgaa cttctctctg ttaaagcaag caggagatgt
     4021 tgaagaaaac cccgggccta tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     4081 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     4141 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     4201 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     4261 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     4321 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     4381 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     4441 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     4501 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     4561 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     4621 ccgcaagccc ggtgccggaa gcggagtgaa acagactttg aattttgacc ttctgaagtt
     4681 ggcaggagac gttgagtcca accctgggcc catggtgagc aagggcgagg agctgttcac
     4741 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4801 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4861 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4921 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4981 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     5041 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     5101 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     5161 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     5221 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     5281 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     5341 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     5401 cactctcggc atggacgagc tgtacaagta aacccagctt tcttgtacaa agtggtgata
     5461 atcgaattcc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     5521 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     5581 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     5641 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     5701 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     5761 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     5821 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt
     5881 tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5941 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     6001 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     6061 gcatcgggaa ttcccgcggt tcgctttaag accaatgact tacaaggcag ctgtagatct
     6121 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     6181 agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag cctgggagct
     6241 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     6301 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     6361 gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc agtatttata
     6421 acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg
     6481 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     6541 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg
     6601 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     6661 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     6721 cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg
     6781 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     6841 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     6901 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     6961 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     7021 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     7081 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     7141 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     7201 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     7261 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     7321 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     7381 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     7441 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     7501 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     7561 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     7621 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     7681 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     7741 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     7801 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     7861 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     7921 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     7981 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     8041 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     8101 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     8161 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     8221 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     8281 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     8341 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     8401 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     8461 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     8521 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     8581 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     8641 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     8701 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     8761 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     8821 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     8881 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     8941 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagagagaaa
     9001 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     9061 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     9121 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     9181 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     9241 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     9301 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     9361 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     9421 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     9481 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     9541 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
     9601 aagggaacaa aagctggagc tgcaagctta a
//
