LOCUS       sequence-472792-       10273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of mRuby2 with 3' microRNA target site only 
            (open-loop).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10273)
  AUTHORS   Love KS, Johnstone CP, Peterman EL, Gaglione S, Birnbaum ME, 
            Galloway KE
  TITLE     Model-guided design of microRNA-based gene circuits supports precise
            dosage of transgenic cargoes into diverse primary cells.
  JOURNAL   Cell Syst. 2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269.
  PUBMED    40300600
REFERENCE   2  (bases 1 to 10273)
  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: "Cell Syst. 
            2025 Apr 22:101269. doi: 10.1016/j.cels.2025.101269."
COMMENT     Sequence Label: 
            pLentiX1-[TRE3G-mRuby2-TS.FF6x1-bGH]-EFS-rtTA-T2A-mGL-P2A-PuroR-WPRE
FEATURES             Location/Qualifiers
     source          1..10273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             88..321
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    393..514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(430..449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     484..503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      554..689
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(709..728)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(710..728)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(738..755)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(738..754)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(747..769)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      896..1351
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(983..1002)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1193..1214
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1377..1481
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1482..1550,1551..2342)
                     /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(1700..1719)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2513..3101
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3002..3021
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3255..3272
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3389..3410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3425..3430,3431..3448,3449..3455)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3463..3479
                     /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     3468..3490
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3487..3503
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3487..3503
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3522..3542
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3524..3542
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3570..3796
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             3797..3977
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    4024..4149
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    4642..4875
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             5060..5104
                     /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    5371..5487
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     polyA_signal    5514..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(5795..6505)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     regulatory      6502..6511
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6566..6944)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    6702..6720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6738..6756
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6774..6792
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6810..6828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6846..6864
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6882..6900
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6918..6936
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(7125..7144)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        7254..7465
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             7478..8221
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             8231..8284
                     /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             join(8285..8287,8288..8290,8291..9001)
                     /codon_start=1
                     /product="monomeric GFP derivative that displays 
                     exceptionally high brightness in cells (Campbell et al., 
                     2020)"
                     /label=mGreenLantern
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLGYGVACFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIVLKGIDFKEDGNILGHKLEYNFNSHKVYITADKQKNGIK
                     ANFKTRHNVEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLK
                     ERVTAAGITHDMDELYK"
     primer_bind     complement(8330..8351)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             9011..9067
                     /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             9068..9670
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGAL"
     primer_bind     complement(9068..9087)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9564..9584
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    9681..10269
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(9734..9754)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10152..10163)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
ORIGIN
        1 cgctgatata gggcccggta cctttaagac caatgactta caaggcagct gtagatctta
       61 gccacttttt aaaagaaaag gggggactgg aagggctagc tcactcccaa cgaagacaag
      121 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      181 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
      241 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
      301 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
      361 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
      421 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      481 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
      541 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
      601 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
      661 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
      721 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
      781 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
      841 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
      901 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
      961 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1021 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1081 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1141 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1201 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1261 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1321 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1381 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1441 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1501 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1561 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1621 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1681 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     1741 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1801 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1861 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1921 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1981 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     2041 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2101 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2161 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2221 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2281 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2341 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2401 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2461 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2521 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2581 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2641 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2701 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2761 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2821 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2881 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2941 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3001 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3061 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3121 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3181 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3241 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     3301 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3361 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     3421 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     3481 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     3541 gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg
     3601 caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
     3661 ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct
     3721 gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct
     3781 agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     3841 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     3901 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     3961 gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag
     4021 gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg
     4081 cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc
     4141 gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg
     4201 ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa
     4261 cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga
     4321 cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta
     4381 gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac
     4441 aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt
     4501 cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt
     4561 agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag
     4621 agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag
     4681 cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat
     4741 agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact
     4801 cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa
     4861 ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt
     4921 gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg
     4981 gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga
     5041 atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag
     5101 tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat
     5161 agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt
     5221 taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga
     5281 caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt
     5341 agtgaacgga tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta
     5401 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     5461 aaaacaaatt acaaaattca aaattttatc gtccagtgtg gtggctacta gcgccataga
     5521 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     5581 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     5641 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     5701 ggggcaaaca acagatggct ggcaactaga aggcacaggt gcaactcctc ctaggtttat
     5761 gaggaatctc tttggttcca caagcttgtt gttacttgta cagctcgtcc atcccaccac
     5821 caagcccggc gaacttggca actgcgtgtt cgcgttgtac tacgaacatt tcattgtcac
     5881 tttcctctaa cctttccagg cggtgatcaa cggcatggat accgggcatc ttgatgttcc
     5941 cgacggtctt ttttgacctg taagttgtta cgaaagagca agacagatgg ccaccaccat
     6001 caactttcag tgccatatga gtgtatcccc tcagaccacc atctgctgga tacatcatct
     6061 ctgtattagg ctcccaaccc ttggtcttct tctgcatcac gggaccattg gagggaaagt
     6121 ttacccctct gacttggacg tggtaaacga gacagccatc ctcaaggctg gtgtcctgca
     6181 tgacggtgac gactccacca tcttcgtatc tcgtaactct ttcccaagta aaaccctcag
     6241 gaaaggactg tttaaagaaa tcaggaatgc ctttcgggta cttgataaaa gtacggctgc
     6301 catacatgaa cgacgtggca agaatgtcaa aggcaaatgg caggggtcct ccctcgatga
     6361 ctttgatcct catggtttga gttcccatgt acggattgcc ttctccttca cctgtgcatt
     6421 tgaattggtg gccgttgacc gaaccttcca tgaccacctt catacgcata ttttccttga
     6481 tcagctcttc gcccttagac accatggtgg ccattacagc gtggatggcg tctccaggcg
     6541 atctgacggt tcactaaacg agctctttac gagggtagga agtggtacgg aaagttggta
     6601 taagacaaaa gtgttgtgga attgctccag gcgatctgac ggttcactaa acgagctctg
     6661 cttttatagg cgcccaccgt acacgcctaa agcttatacg ttctctatca ctgataggga
     6721 gtaaactgga tatacgttct ctatcactga tagggagtaa actgtagata cgttctctat
     6781 cactgatagg gagtaaactg gtcatacgtt ctctatcact gatagggagt aaactcctta
     6841 tacgttctct atcactgata gggagtaaag tctgcatacg ttctctatca ctgataggga
     6901 gtaaactctt catacgttct ctatcactga tagggagtaa actcgaggaa tagtacgaac
     6961 atggtagcta gtggatcctt cgtcgacctg cagactggct gtgtataagg gagcctgaca
     7021 tttatattcc ccagaacatc aggttaatgg cgtttttgat gtcattttcg cggtggctga
     7081 gatcagccac ttcttccccg ataacggaga ccggcacact ggcgcatatc ggtggtcatc
     7141 atgcgccagc tttcatcccc gatatgcacc accgggtaaa gttcacgcat atgaagctgg
     7201 acatcacctc ccacaacctc gaggtggaac agtacgctga tatctcttat actgggcaga
     7261 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
     7321 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
     7381 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
     7441 caacgggttt gccgccagaa cacagtaatg tgccaccatg agccggctgg acaagagcaa
     7501 agtgatcaac agcgccctgg aactgctgaa cggcgtgggc atcgagggcc tgaccacccg
     7561 gaagctggcc cagaaactgg gcgtggaaca gcccaccctg tactggcacg tgaagaacaa
     7621 gcgggccctg ctggacgccc tgcccatcga gatgctggac cggcaccaca cccacagctg
     7681 ccctctggaa ggcgagagct ggcaggactt cctgcggaac aacgccaaga gctacagatg
     7741 cgccctgctg agccaccggg acggcgccaa agtgcacctg ggcaccagac ccaccgagaa
     7801 gcagtacgag acactggaaa accagctggc cttcctgtgc cagcagggct tcagcctgga
     7861 aaacgccctg tacgccctga gcgccgtggg ccacttcacc ctgggctgcg tgctggaaga
     7921 acaggaacac caggtcgcca aagaggaaag agagacaccc accaccgaca gcatgccccc
     7981 cctgctgaag caggccatcg agctgttcga cagacagggc gccgagcccg ccttcctgtt
     8041 cggcctggaa ctgatcatct gcggcctcga gaagcagctg aagtgcgaga gcggcggacc
     8101 caccgacgcc ctggacgact tcgacctgga catgctgccc gccgatgccc tggatgattt
     8161 tgatctggat atgctccctg ctgacgctct cgatgacttc gatctcgata tgctgccagg
     8221 cggaagcgga gaaggtagag gttctctcct cacttgtggt gatgttgaag aaaaccctgg
     8281 tccaatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     8341 ggacggcgac gtaaacggcc acaagttcag cgtccgcggc gagggcgagg gcgatgccac
     8401 caacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     8461 caccctcgtg accaccttag gctacggcgt ggcctgcttc gcccgctacc ccgaccacat
     8521 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     8581 ctctttcaag gacgacggta cctacaagac ccgcgccgag gtgaagttcg agggcgacac
     8641 cctggtgaac cgcatcgtgc tgaagggcat cgacttcaag gaggacggca acatcctggg
     8701 gcacaagctg gagtacaact tcaacagcca caaggtctat atcacggccg acaagcagaa
     8761 gaacggcatc aaggctaact tcaagacccg ccacaacgtt gaggacggcg gcgtgcagct
     8821 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     8881 ccactacctg agccatcagt ccaaactgag caaagacccc aacgagaagc gcgatcacat
     8941 ggtcctgaag gagagggtga ccgccgccgg gattacacat gacatggacg agctgtacaa
     9001 gggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
     9061 tggacctatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     9121 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     9181 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     9241 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     9301 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     9361 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     9421 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     9481 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     9541 cttcctggag acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     9601 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     9661 tgccttgtaa caacgccgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     9721 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     9781 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     9841 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     9901 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     9961 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
    10021 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
    10081 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
    10141 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
    10201 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
    10261 cctccccgcc gct
//
