LOCUS       sequence-472756-       10648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of mRuby2 with intronic microRNA only.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10648)
  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 10648)
  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(miRE.FF4)-bGH]-EFS-rtTA-T2A-mGL-P2A-PuroR-WPR
            E
FEATURES             Location/Qualifiers
     source          1..10648
                     /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"
     ncRNA           complement(6459..6571)
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           complement(6628..6642)
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     regulatory      6877..6886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6941..7319)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    7077..7095
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7113..7131
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7149..7167
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7185..7203
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7221..7239
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7257..7275
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7293..7311
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(7500..7519)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        7629..7840
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             7853..8596
                     /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             8606..8659
                     /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(8660..8662,8663..8665,8666..9376)
                     /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(8705..8726)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             9386..9442
                     /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             9443..10045
                     /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(9443..9462)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9939..9959
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10056..10644
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10109..10129)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10527..10538)
                     /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 gcaacgttgt tacttgtaca
     5761 gctcgtccat cccaccacca agcccggcga acttggcaac tgcgtgttcg cgttgtacta
     5821 cgaacatttc attgtcactt tcctctaacc tttccaggcg gtgatcaacg gcatggatac
     5881 cgggcatctt gatgttcccg acggtctttt ttgacctgta agttgttacg aaagagcaag
     5941 acagatggcc accaccatca actttcagtg ccatatgagt gtatcccctc agaccaccat
     6001 ctgctggata catcatctct gtattaggct cccaaccctt ggtcttcttc tgcatcacgg
     6061 gaccattgga gggaaagttt acccctctga cttggacgtg gtaaacgaga cagccatcct
     6121 caaggctggt gtcctgcatg acggtgacga ctccaccatc ttcgtatctc gtaactcttt
     6181 cccaagtaaa accctcagga aaggactgtt taaagaaatc aggaatgcct ttcgggtact
     6241 tgataaaagt acggctgcca tacatgaacg acgtggcaag aatgtcaaag gcaaatggca
     6301 ggggtcctcc ctcgatgact ttgatcctca tggtttgagt tcccatgtac ggattgcctt
     6361 ctccttcacc tgtgcatttg aattggtggc cgttgaccga accttccatg accacctgtg
     6421 aaaaaaaaaa agaagactcg aagttagtac aattgaaaaa agtgatttaa tttataccat
     6481 tttaattcag ctttgtaaaa atgtatcaaa gagatagcaa ggtattcagt tttagtaaac
     6541 aagataattg ctcctaaagt agccccttga attctagccc cttgaagtcc gaggcagtag
     6601 gcaccgcttg aagtctttaa ttaaatacat ctgtggcttc actatttaat taaagacttc
     6661 aagcggcgct cactgtcaac agcaatatac cttctcgagc cttctgttgg gttaagaagt
     6721 ccctgaagaa gtaatcccag caagtgtttc caagatgtgc aggcaacgat tctgtaaagt
     6781 actgaagcct cattcaacat agtatatgtg ctgccgaagc gagcacactt accttcatac
     6841 gcatattttc cttgatcagc tcttcgccct tagacaccat ggtggccatt acagcgtgga
     6901 tggcgtctcc aggcgatctg acggttcact aaacgagctc tttacgaggg taggaagtgg
     6961 tacggaaagt tggtataaga caaaagtgtt gtggaattgc tccaggcgat ctgacggttc
     7021 actaaacgag ctctgctttt ataggcgccc accgtacacg cctaaagctt atacgttctc
     7081 tatcactgat agggagtaaa ctggatatac gttctctatc actgataggg agtaaactgt
     7141 agatacgttc tctatcactg atagggagta aactggtcat acgttctcta tcactgatag
     7201 ggagtaaact ccttatacgt tctctatcac tgatagggag taaagtctgc atacgttctc
     7261 tatcactgat agggagtaaa ctcttcatac gttctctatc actgataggg agtaaactcg
     7321 aggaatagta cgaacatggt agctagtgga tccttcgtcg acctgcagac tggctgtgta
     7381 taagggagcc tgacatttat attccccaga acatcaggtt aatggcgttt ttgatgtcat
     7441 tttcgcggtg gctgagatca gccacttctt ccccgataac ggagaccggc acactggcgc
     7501 atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca
     7561 cgcatatgaa gctggacatc acctcccaca acctcgaggt ggaacagtac gctgatatct
     7621 cttatactgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg
     7681 caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta
     7741 ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt
     7801 gaacgttctt tttcgcaacg ggtttgccgc cagaacacag taatgtgcca ccatgagccg
     7861 gctggacaag agcaaagtga tcaacagcgc cctggaactg ctgaacggcg tgggcatcga
     7921 gggcctgacc acccggaagc tggcccagaa actgggcgtg gaacagccca ccctgtactg
     7981 gcacgtgaag aacaagcggg ccctgctgga cgccctgccc atcgagatgc tggaccggca
     8041 ccacacccac agctgccctc tggaaggcga gagctggcag gacttcctgc ggaacaacgc
     8101 caagagctac agatgcgccc tgctgagcca ccgggacggc gccaaagtgc acctgggcac
     8161 cagacccacc gagaagcagt acgagacact ggaaaaccag ctggccttcc tgtgccagca
     8221 gggcttcagc ctggaaaacg ccctgtacgc cctgagcgcc gtgggccact tcaccctggg
     8281 ctgcgtgctg gaagaacagg aacaccaggt cgccaaagag gaaagagaga cacccaccac
     8341 cgacagcatg ccccccctgc tgaagcaggc catcgagctg ttcgacagac agggcgccga
     8401 gcccgccttc ctgttcggcc tggaactgat catctgcggc ctcgagaagc agctgaagtg
     8461 cgagagcggc ggacccaccg acgccctgga cgacttcgac ctggacatgc tgcccgccga
     8521 tgccctggat gattttgatc tggatatgct ccctgctgac gctctcgatg acttcgatct
     8581 cgatatgctg ccaggcggaa gcggagaagg tagaggttct ctcctcactt gtggtgatgt
     8641 tgaagaaaac cctggtccaa tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     8701 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtcc gcggcgaggg
     8761 cgagggcgat gccaccaacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     8821 gcccgtgccc tggcccaccc tcgtgaccac cttaggctac ggcgtggcct gcttcgcccg
     8881 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     8941 ccaggagcgc accatctctt tcaaggacga cggtacctac aagacccgcg ccgaggtgaa
     9001 gttcgagggc gacaccctgg tgaaccgcat cgtgctgaag ggcatcgact tcaaggagga
     9061 cggcaacatc ctggggcaca agctggagta caacttcaac agccacaagg tctatatcac
     9121 ggccgacaag cagaagaacg gcatcaaggc taacttcaag acccgccaca acgttgagga
     9181 cggcggcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt
     9241 gctgctgccc gacaaccact acctgagcca tcagtccaaa ctgagcaaag accccaacga
     9301 gaagcgcgat cacatggtcc tgaaggagag ggtgaccgcc gccgggatta cacatgacat
     9361 ggacgagctg tacaagggaa gcggagctac taacttcagc ctgctgaagc aggctggaga
     9421 cgtggaggag aaccctggac ctatgaccga gtacaagccc acggtgcgcc tcgccacccg
     9481 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     9541 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     9601 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     9661 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     9721 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     9781 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     9841 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     9901 cgccggggtg cccgccttcc tggagacatc cgcgccccgc aacctcccct tctacgagcg
     9961 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
    10021 gacccgcaag cccggtgcct tgtaacaacg ccgataatca acctctggat tacaaaattt
    10081 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
    10141 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
    10201 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
    10261 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
    10321 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
    10381 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
    10441 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
    10501 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
    10561 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
    10621 tctccctttg ggccgcctcc ccgccgct
//
