LOCUS       sequence-263335-        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        3..229
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             230..410
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    457..582
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1075..1308
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1493..1537
                     /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    1803..1920
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    1938..1958
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1962..2462
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2381..2399
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    2463..2487
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2494..3516
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2494..3516
                     /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             3526..3579
                     /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             3580..3975
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             3985..4041
                     /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             4042..4638
                     /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(4042..4061)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4538..4558
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4648..4713
                     /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(4714..4716,4717..4719,4720..5433)
                     /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(4714..4716,4717..4719,4720..5430)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4759..4780)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5020..5039)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5367..5388
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5434..5458)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    5476..6064
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5529..5549)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5947..5958)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6152..6385
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6457..6578
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6494..6513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6548..6567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6618..6753
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6773..6792)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6774..6792)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6802..6819)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6802..6818)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6811..6833)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6960..7415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7047..7066)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7257..7278
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7441..7545
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7546..7614,7615..8406)
                     /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(7764..7783)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8577..9165
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9066..9085
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9319..9336
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9453..9474
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9489..9494,9495..9512,9513..9519)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9527..9543
                     /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     9532..9554
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9551..9567
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9551..9567
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9586..9606
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        9588..9606
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca
       61 tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga
      121 tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt
      181 gccgcattgc agagatattg tatttaagtg cctagctcga tacataaacg ggtctctctg
      241 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
      301 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
      361 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg
      421 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt
      481 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg
      541 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga
      601 attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta
      661 aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta
      721 gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga
      781 tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg
      841 atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt
      901 aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga
      961 caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc
     1021 acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc
     1081 tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct
     1141 gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag
     1201 ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca
     1261 ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg
     1321 ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa
     1381 atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgcta
     1801 gcttttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata
     1861 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt
     1921 actagtgatt atcggatcaa ctttgtatag aaaagttggg gttgcgcctt ttccaaggca
     1981 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     2041 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     2101 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     2161 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
     2221 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     2281 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     2341 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     2401 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     2461 aggcaagttt gtacaaaaaa gcaggctgcc accatgaaaa agcctgaact caccgcgacg
     2521 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     2581 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     2641 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     2701 gccgcgctcc cgattccgga agtgcttgac attggggaat ttagcgagag cctgacctat
     2761 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     2821 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     2881 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     2941 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3001 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3061 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3121 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     3181 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     3241 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     3301 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     3361 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     3421 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     3481 aaccgacgcc ccagcactcg tccgagggca aaggaaggaa gcggagaggg caggggaagt
     3541 cttctaacat gcggggacgt ggaggaaaat cccggcccca tggccaagcc tttgtctcaa
     3601 gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc catctctgaa
     3661 gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac tggtgtcaat
     3721 gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac tgctgctgct
     3781 gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag gggcatcttg
     3841 agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat caaagccata
     3901 gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct gccctctggt
     3961 tatgtgtggg agggcggaag cggagccacg aacttctctc tgttaaagca agcaggagat
     4021 gttgaagaaa accccgggcc tatgaccgag tacaagccca cggtgcgcct cgccacccgc
     4081 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     4141 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     4201 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     4261 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     4321 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     4381 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     4441 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     4501 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     4561 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     4621 acccgcaagc ccggtgccgg aagcggagtg aaacagactt tgaattttga ccttctgaag
     4681 ttggcaggag acgttgagtc caaccctggg cccatggtga gcaagggcga ggagctgttc
     4741 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     4801 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     4861 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     4921 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     4981 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     5041 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     5101 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     5161 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     5221 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     5281 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     5341 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     5401 atcactctcg gcatggacga gctgtacaag taaacccagc tttcttgtac aaagtggtga
     5461 taatcgaatt ccgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     5521 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     5581 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     5641 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     5701 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     5761 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     5821 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc
     5881 tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     5941 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     6001 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     6061 ccgcatcggg aattcccgcg gttcgcttta agaccaatga cttacaaggc agctgtagat
     6121 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     6181 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     6241 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     6301 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     6361 tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat tcagtattta
     6421 taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc agcttataat
     6481 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     6541 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct ctagctatcc
     6601 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     6661 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     6721 tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt cgccctatag
     6781 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6841 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6901 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga
     6961 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     7021 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     7081 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     7141 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     7201 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     7261 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     7321 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     7381 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     7441 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     7501 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     7561 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     7621 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     7681 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     7741 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     7801 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     7861 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     7921 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     7981 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     8041 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     8101 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     8161 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     8221 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     8281 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     8341 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     8401 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     8461 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     8521 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     8581 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     8641 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     8701 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     8761 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     8821 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     8881 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     8941 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagagaga
     9001 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     9061 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     9121 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     9181 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     9241 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     9301 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
     9361 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     9421 gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca
     9481 ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa
     9541 caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat taaccctcac
     9601 taaagggaac aaaagctgga gctgcaagct t
//
