LOCUS       sequence-455149-       12837 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Landing pad with Bxb1 attP_wildtype site, Dox-inducible 
            BFP-iCasp9-Blasticidin, CMV-driven rtTA3, T2A-neomycin for HDR into 
            AAVS1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12837)
  AUTHORS   Nugent PJ, Park H, Wladyka CL, Chen KY, Bynum C, Quarterman G, Hsieh
            AC, Subramaniam AR
  TITLE     Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human 
            Cells.
  JOURNAL   bioRxiv [Preprint]. 2024 Jul 26:2024.07.25.605204. doi: 
            10.1101/2024.07.25.605204.
  PUBMED    39091804
REFERENCE   2  (bases 1 to 12837)
  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: "bioRxiv 
            [Preprint]. 2024 Jul 26:2024.07.25.605204. doi: 
            10.1101/2024.07.25.605204."
COMMENT     Sequence Label: pHPHS232
FEATURES             Location/Qualifiers
     source          1..12837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(103..122)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             114..923
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(181..200)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1101..1689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1590..1609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1843..1860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1977..1998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2013..2018,2019..2036,2037..2043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2051..2067
                     /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     2056..2078
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2075..2091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2075..2091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    2112..2915
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    complement(2924..2957)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2958..2983
                     /label=SA
                     /note="splice acceptor site"
     CDS             3007..3060
                     /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             3055..3855
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="GPRIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3115..3134)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3725..3744
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3890..4114
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(4115..4148)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        5399..5748
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    5407..5425
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5443..5461
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5479..5497
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5515..5533
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5551..5569
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5587..5605
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5623..5641
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5750..5797
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     regulatory      5798..5807
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5804..6514
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             6524..6580
                     /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             6596..6916
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             complement(7268..7279)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             7793..7819
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             7838..7891
                     /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             7892..8287
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="AKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGV
                     NVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIK
                     AIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(8380..8399)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    8445..8500
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(8499..8518)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     protein_bind    8833..8866
                     /label=loxP511
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATAC) (Shaw et al., 2021). loxP511 sites are 
                     compatible with each other, but incompatible with wild-type
                     loxP sites."
     enhancer        10121..10424
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10425..10628
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    10733..10757
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             10760..11467
                     /codon_start=1
                     /product="reverse tetracycline transactivator 3"
                     /label=rtTA3
                     /note="activates expression from Tet-responsive promoters 
                     in the presence of doxycycline"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPG"
     primer_bind     complement(11492..11511)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     11546..11565
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    11598..12434
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(12443..12460)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(12443..12459)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(12452..12474)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(12668..12687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     12787..12809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tgcagctctg gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaatatat
       61 catcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc
      121 atattcaacg ggaaacgtcg aggccgcgat taaattccaa catggatgct gatttatatg
      181 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgcttgtatg
      241 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
      301 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
      361 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggaaaaa
      421 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
      481 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
      541 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
      601 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
      661 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
      721 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
      781 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
      841 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
      901 tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacat tattcagatt
      961 gggcttgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1021 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     1141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     1201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     1261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     1321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     1381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     1441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     1501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     1561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     1621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     1681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     1741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     1801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     1861 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1921 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     1981 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     2041 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     2101 gccgcggccg ctgctttctc tgaccagcat tctctcccct gggcctgtgc cgctttctgt
     2161 ctgcagcttg tggcctgggt cacctctacg gctggcccag atccttccct gccgcctcct
     2221 tcaggttccg tcttcctcca ctccctcttc cccttgctct ctgctgtgtt gctgcccaag
     2281 gatgctcttt ccggagcact tccttctcgg cgctgcacca cgtgatgtcc tctgagcgga
     2341 tcctccccgt gtctgggtcc tctccgggca tctctcctcc ctcacccaac cccatgccgt
     2401 cttcactcgc tgggttccct tttccttctc cttctggggc ctgtgccatc tctcgtttct
     2461 taggatggcc ttctccgacg gatgtctccc ttgcgtcccg cctccccttc ttgtaggcct
     2521 gcatcatcac cgtttttctg gacaacccca aagtaccccg tctccctggc tttagccacc
     2581 tctccatcct cttgctttct ttgcctggac accccgttct cctgtggatt cgggtcacct
     2641 ctcactcctt tcatttgggc agctccccta ccccccttac ctctctagtc tgtgctagct
     2701 cttccagccc cctgtcatgg catcttccag gggtccgaga gctcagctag tcttcttcct
     2761 ccaacccggg cccctatgtc cacttcagga cagcatgttt gctgcctcca gggatcctgt
     2821 gtccccgagc tgggaccacc ttatattccc agggccggtt aatgtggctc tggttctggg
     2881 tacttttatc tgtcccctcc accccacagt ggggcgcggc cgcataactt cgtatagcat
     2941 acattatacg aagttatctg acctcttctc ttcctcccac agggcctcga gagatctggc
     3001 agcggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     3061 aggattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3121 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3181 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3241 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3301 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3361 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     3421 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     3481 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     3541 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     3601 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     3661 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     3721 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     3781 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     3841 gacgagttct tctaacacct ggtgcatgac ccgcaagccc ggtgcctgac tgtgccttct
     3901 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3961 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     4021 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     4081 agcaggcatg ctggggatgc ggtgggctct atggataact tcgtatagca tacattatac
     4141 gaagttatga gctcacgggg acagcccccc cccaaagccc ccagggatgt aattacgtcc
     4201 ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc
     4261 ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga
     4321 tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg
     4381 gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg
     4441 ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac
     4501 cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg
     4561 gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact
     4621 gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc
     4681 tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca
     4741 gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga
     4801 catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa
     4861 ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg gagaggagca
     4921 caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg
     4981 gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt cagtaggact
     5041 gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa tcatgaaggc
     5101 tggaaaagcc ctccaagatc cccaagacca accccaaccc acccaccgtg cccactggcc
     5161 atgtccctca gtgccacatc cccacagttc ttcatcacct ccagggacgg tgaccccccc
     5221 acctccgtgg gcagctgtgc cactgcagca ccgctctttg gagaaggtaa atcttgctaa
     5281 atccagcccg accctcccct ggcacaacgt aaggccatta tctctcatcc aactccagga
     5341 cggagtcagt gagaatattg gcgcgccgat atccgatgag gccctttcgt cttcactcga
     5401 gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc agtgatagag
     5461 aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt ttactcccta
     5521 tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa cgtatctaca
     5581 gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc agtgatagag
     5641 aacgtatgtc gaggtaggcg tgtacggtgg gcgcctataa aagcagagct cgtttagtga
     5701 accgtcagat cgcctggagc aattccacaa cacttttgtc ttatacttgg gtttgtctgg
     5761 tcaaccaccg cggtctcagt ggtgtacggt acaaaccgcc accatggtgt ctaagggcga
     5821 agagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg tggacaacca
     5881 tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag
     5941 aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctactagctt
     6001 cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact tcttcaagca
     6061 gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct
     6121 gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg tcaagatcag
     6181 aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg gctgggaggc
     6241 cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg acatggccct
     6301 gaagctcgtg ggcgggagcc atctgatcgc aaacgccaag accacatata gatccaagaa
     6361 acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac tggaaagaat
     6421 caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg ccagatactg
     6481 cgacctccct agcaaactgg ggcacaagct taatgggagc ggagctacta acttcagcct
     6541 gctgaagcag gctggagacg tggaggagaa ccctggacct tccgccaacc tcgagggagt
     6601 gcaggtggag actatctccc caggagacgg gcgcaccttc cccaagcgcg gccagacctg
     6661 cgtggtgcac tacaccggga tgcttgaaga tggaaagaaa gttgattcct cccgggacag
     6721 aaacaagccc tttaagttta tgctaggcaa gcaggaggtg atccgaggct gggaagaagg
     6781 ggttgcccag atgagtgtgg gtcagagagc caaactgact atatctccag attatgccta
     6841 tggtgccact gggcacccag gcatcatccc accacatgcc actctcgtct tcgatgtgga
     6901 gcttctaaaa ctggaatctg gcggtggatc cggagtcgac ggatttggtg atgtcggtgc
     6961 tcttgagagt ttgaggggaa atgcagattt ggcttacatc ctgagcatgg agccctgtgg
     7021 ccactgcctc attatcaaca atgtgaactt ctgccgtgag tccgggctcc gcacccgcac
     7081 tggctccaac atcgactgtg agaagttgcg gcgtcgcttc tcctcgctgc atttcatggt
     7141 ggaggtgaag ggcgacctga ctgccaagaa aatggtgctg gctttgctgg agctggcgcg
     7201 gcaggaccac ggtgctctgg actgctgcgt ggtggtcatt ctctctcacg gctgtcaggc
     7261 cagccacctg cagttcccag gggctgtcta cggcacagat ggatgccctg tgtcggtcga
     7321 gaagattgtg aacatcttca atgggaccag ctgccccagc ctgggaggga agcccaagct
     7381 ctttttcatc caggcctgtg gtggggagca gaaagaccat gggtttgagg tggcctccac
     7441 ttcccctgaa gacgagtccc ctggcagtaa ccccgagcca gatgccaccc cgttccagga
     7501 aggtttgagg accttcgacc agctggacgc catatctagt ttgcccacac ccagtgacat
     7561 ctttgtgtcc tactctactt tcccaggttt tgtttcctgg agggacccca agagtggctc
     7621 ctggtacgtt gagaccctgg acgacatctt tgagcagtgg gctcactctg aagacctgca
     7681 gtccctcctg cttagggtcg ctaatgctgt ttcggtgaaa gggatttata aacagatgcc
     7741 tggttgcttt aatttcctcc ggaaaaaact tttctttaaa acatcagtcg actatccgta
     7801 cgacgtacca gactacgcac tcgactctgg cagcggagag ggcagaggaa gtcttctaac
     7861 atgcggtgac gtggaggaga atcccggccc tgccaagcct ttgtctcaag aagaatccac
     7921 cctcattgaa agagcaacgg ctacaatcaa cagcatcccc atctctgaag actacagcgt
     7981 cgccagcgca gctctctcta gcgacggccg catcttcact ggtgtcaatg tatatcattt
     8041 tactggggga ccttgtgcag aactcgtggt gctgggcact gctgctgctg cggcagctgg
     8101 caacctgact tgtatcgtcg cgatcggaaa tgagaacagg ggcatcttga gcccctgcgg
     8161 acggtgccga caggtgcttc tcgatctgca tcctgggatc aaagccatag tgaaggacag
     8221 tgatggacag ccgacggcag ttgggattcg tgaattgctg ccctctggtt atgtgtggga
     8281 gggctagtaa acgcgtgaat tcactcctca ggtgcaggct gcctatcaga aggtggtggc
     8341 tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa
     8401 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     8461 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     8521 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     8581 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc
     8641 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     8701 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     8761 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     8821 gagatcaccg gtataacttc gtataatgta tactatacga agttatggta ccttaattaa
     8881 gttaacatgc atagtactga gctcacgggg acagcccccc cccaaagccc ccagggatgt
     8941 aattacgtcc ctcccccgct agggggcagc agcgagccgc ccggggctcc gctccggtcc
     9001 ggcgctcccc ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg
     9061 tggcacggga tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg
     9121 gggggatacg gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga
     9181 acggcctggg ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag
     9241 ggtcatcaac cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg
     9301 cagggatggg gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg
     9361 gggaaaaact gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc
     9421 ccccttgtcc tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt
     9481 tgccaattca gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat
     9541 ggacgtggga catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga
     9601 acagccttaa ggataagaag ataggataga aggacaaaga gcaagttaaa acccagcatg
     9661 gagaggagca caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg
     9721 atggtgtctg gatgcaagca gaaggggtgg aagagcttgc ctggagagat acagctgggt
     9781 cagtaggact gggacaggca gctggagaat tgccatgtag atgttcatac aatcgtcaaa
     9841 tcatgaaggc tggaaaagcc ctccaagatc cccaagacca accccaaccc acccaccgtg
     9901 cccactggcc atgtccctca gtgccacatc cccacagttc ttcatcacct ccagggacgg
     9961 tgaccccccc acctccgtgg gcagctgtgc cactgcagca ccgctctttg gagaaggtaa
    10021 atcttgctaa atccagcccg accctcccct ggcacaacgt aaggccatta tctctcatcc
    10081 aactccagga cggagtcagt gagaatattg tttaaacccg cgttacataa cttacggtaa
    10141 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
    10201 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
    10261 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
    10321 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
    10381 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
    10441 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
    10501 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
    10561 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
    10621 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
    10681 tccatagaag acaccgactc tagtccagtg tggtggaatt ctgcagatat caacaagttt
    10741 gtacaaaaaa gttggcacca tgtctagact ggacaagagc aaagtcataa acggagctct
    10801 ggaattactc aatggtgtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct
    10861 gggagttgag cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc
    10921 cctgccaatc gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc
    10981 atggcaagac tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg
    11041 cgacggggct aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga
    11101 aaatcagctc gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct
    11161 gtccgccgtg ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc
    11221 aaaagaggaa agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat
    11281 tgagctgttc gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat
    11341 atgtggcctg gagaaacagc taaagtgcga aagcggcggg ccgaccgacg cccttgacga
    11401 ttttgactta gacatgctcc cagccgatgc ccttgacgat tttgaccttg acatgctccc
    11461 cgggtaagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
    11521 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
    11581 tataagctgc aataaacact agggacagga ttggtgacag aaaagcccca tccttaggcc
    11641 tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag gcagattcct
    11701 tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga acctctaagg
    11761 tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg gggtgggagg
    11821 gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta ccctctccca
    11881 gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct
    11941 tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat aagttggtcc
    12001 tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt cctccgtgcg
    12061 tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg ctgtggtgag
    12121 gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc
    12181 aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt aaagagtccc
    12241 cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc taaggccctg
    12301 ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag gcttccctgt
    12361 cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc tacaggggtt
    12421 cctggctctg ctctgtttaa acactggccg tcgttttaca acgtcgtgac tgggaaaacc
    12481 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
    12541 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
    12601 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
    12661 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
    12721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
    12781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcga
//
