LOCUS       sequence-472790-       10656 bp    DNA     circular SYN 14-MAY-2025
DEFINITION  Lentiviral expression of mRuby2 with 3' microRNA only.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10656)
  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 10656)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 14, 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-WPRE
FEATURES             Location/Qualifiers
     source          1..10656
                     /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(5795..5907)
                     /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(5964..5978)
                     /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"
     CDS             complement(6178..6888)
                     /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      6885..6894
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6949..7327)
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    7085..7103
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7121..7139
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7157..7175
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7193..7211
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7229..7247
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7265..7283
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7301..7319
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     complement(7508..7527)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     promoter        7637..7848
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             7861..8604
                     /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             8614..8667
                     /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(8668..8670,8671..8673,8674..9384)
                     /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(8713..8734)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             9394..9450
                     /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             9451..10053
                     /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(9451..9470)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     9947..9967
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    10064..10652
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(10117..10137)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(10535..10546)
                     /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 gcaactccta cctgtgaaaa
     5761 aaaaaaagaa gactcgaagt tagtacaatt gaaaaaagtg atttaattta taccatttta
     5821 attcagcttt gtaaaaatgt atcaaagaga tagcaaggta ttcagtttta gtaaacaaga
     5881 taattgctcc taaagtagcc ccttgaattc tagccccttg aagtccgagg cagtaggcac
     5941 cgcttgaagt ctttaattaa atacatctgt ggcttcacta tttaattaaa gacttcaagc
     6001 ggcgctcact gtcaacagca atataccttc tcgagccttc tgttgggtta agaagtccct
     6061 gaagaagtaa tcccagcaag tgtttccaag atgtgcaggc aacgattctg taaagtactg
     6121 aagcctcatt caacatagta tatgtgctgc cgaagcgagc acacttacct gttgttactt
     6181 gtacagctcg tccatcccac caccaagccc ggcgaacttg gcaactgcgt gttcgcgttg
     6241 tactacgaac atttcattgt cactttcctc taacctttcc aggcggtgat caacggcatg
     6301 gataccgggc atcttgatgt tcccgacggt cttttttgac ctgtaagttg ttacgaaaga
     6361 gcaagacaga tggccaccac catcaacttt cagtgccata tgagtgtatc ccctcagacc
     6421 accatctgct ggatacatca tctctgtatt aggctcccaa cccttggtct tcttctgcat
     6481 cacgggacca ttggagggaa agtttacccc tctgacttgg acgtggtaaa cgagacagcc
     6541 atcctcaagg ctggtgtcct gcatgacggt gacgactcca ccatcttcgt atctcgtaac
     6601 tctttcccaa gtaaaaccct caggaaagga ctgtttaaag aaatcaggaa tgcctttcgg
     6661 gtacttgata aaagtacggc tgccatacat gaacgacgtg gcaagaatgt caaaggcaaa
     6721 tggcaggggt cctccctcga tgactttgat cctcatggtt tgagttccca tgtacggatt
     6781 gccttctcct tcacctgtgc atttgaattg gtggccgttg accgaacctt ccatgaccac
     6841 cttcatacgc atattttcct tgatcagctc ttcgccctta gacaccatgg tggccattac
     6901 agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctctt tacgagggta
     6961 ggaagtggta cggaaagttg gtataagaca aaagtgttgt ggaattgctc caggcgatct
     7021 gacggttcac taaacgagct ctgcttttat aggcgcccac cgtacacgcc taaagcttat
     7081 acgttctcta tcactgatag ggagtaaact ggatatacgt tctctatcac tgatagggag
     7141 taaactgtag atacgttctc tatcactgat agggagtaaa ctggtcatac gttctctatc
     7201 actgataggg agtaaactcc ttatacgttc tctatcactg atagggagta aagtctgcat
     7261 acgttctcta tcactgatag ggagtaaact cttcatacgt tctctatcac tgatagggag
     7321 taaactcgag gaatagtacg aacatggtag ctagtggatc cttcgtcgac ctgcagactg
     7381 gctgtgtata agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt
     7441 gatgtcattt tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac
     7501 actggcgcat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt
     7561 aaagttcacg catatgaagc tggacatcac ctcccacaac ctcgaggtgg aacagtacgc
     7621 tgatatctct tatactgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga
     7681 ggggtcggca attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat
     7741 gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta
     7801 gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagta atgtgccacc
     7861 atgagccggc tggacaagag caaagtgatc aacagcgccc tggaactgct gaacggcgtg
     7921 ggcatcgagg gcctgaccac ccggaagctg gcccagaaac tgggcgtgga acagcccacc
     7981 ctgtactggc acgtgaagaa caagcgggcc ctgctggacg ccctgcccat cgagatgctg
     8041 gaccggcacc acacccacag ctgccctctg gaaggcgaga gctggcagga cttcctgcgg
     8101 aacaacgcca agagctacag atgcgccctg ctgagccacc gggacggcgc caaagtgcac
     8161 ctgggcacca gacccaccga gaagcagtac gagacactgg aaaaccagct ggccttcctg
     8221 tgccagcagg gcttcagcct ggaaaacgcc ctgtacgccc tgagcgccgt gggccacttc
     8281 accctgggct gcgtgctgga agaacaggaa caccaggtcg ccaaagagga aagagagaca
     8341 cccaccaccg acagcatgcc ccccctgctg aagcaggcca tcgagctgtt cgacagacag
     8401 ggcgccgagc ccgccttcct gttcggcctg gaactgatca tctgcggcct cgagaagcag
     8461 ctgaagtgcg agagcggcgg acccaccgac gccctggacg acttcgacct ggacatgctg
     8521 cccgccgatg ccctggatga ttttgatctg gatatgctcc ctgctgacgc tctcgatgac
     8581 ttcgatctcg atatgctgcc aggcggaagc ggagaaggta gaggttctct cctcacttgt
     8641 ggtgatgttg aagaaaaccc tggtccaatg gtgagcaagg gcgaggagct gttcaccggg
     8701 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtccgc
     8761 ggcgagggcg agggcgatgc caccaacggc aagctgaccc tgaagttcat ctgcaccacc
     8821 ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct taggctacgg cgtggcctgc
     8881 ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     8941 ggctacgtcc aggagcgcac catctctttc aaggacgacg gtacctacaa gacccgcgcc
     9001 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg tgctgaaggg catcgacttc
     9061 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaaggtc
     9121 tatatcacgg ccgacaagca gaagaacggc atcaaggcta acttcaagac ccgccacaac
     9181 gttgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     9241 ggccccgtgc tgctgcccga caaccactac ctgagccatc agtccaaact gagcaaagac
     9301 cccaacgaga agcgcgatca catggtcctg aaggagaggg tgaccgccgc cgggattaca
     9361 catgacatgg acgagctgta caagggaagc ggagctacta acttcagcct gctgaagcag
     9421 gctggagacg tggaggagaa ccctggacct atgaccgagt acaagcccac ggtgcgcctc
     9481 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     9541 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     9601 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     9661 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     9721 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     9781 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     9841 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     9901 gccgagcgcg ccggggtgcc cgccttcctg gagacatccg cgccccgcaa cctccccttc
     9961 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
    10021 tggtgcatga cccgcaagcc cggtgccttg taacaacgcc gataatcaac ctctggatta
    10081 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
    10141 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
    10201 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
    10261 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
    10321 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
    10381 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
    10441 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
    10501 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
    10561 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
    10621 gagtcggatc tccctttggg ccgcctcccc gccgct
//
