LOCUS       sequence-358738-       13898 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral vector for dox-inducible expression of APOBEC1-YTH in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13898)
  AUTHORS   Tegowski M, Flamand MN, Meyer KD
  TITLE     scDART-seq reveals distinct m(6)A signatures and mRNA methylation 
            heterogeneity in single cells.
  JOURNAL   Mol Cell. 2022 Feb 17;82(4):868-878.e10. doi: 
            10.1016/j.molcel.2021.12.038. Epub 2022 Jan 25.
  PUBMED    35081365
REFERENCE   2  (bases 1 to 13898)
  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"; doi: 
            "10.1016/j.molcel.2021.12.038"; journalName: "Mol Cell"; date: 
            "2022-02-17- 17"; volume: "82"; issue: "4"; pages: "868-878.e10"
COMMENT     Sequence Label: TLCV2-APOBEC1-YTH
FEATURES             Location/Qualifiers
     source          1..13898
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        238..313
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        350..664
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    358..376
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    394..412
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    429..447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    465..483
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    501..519
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    536..554
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    572..590
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     637..661
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             696..1382
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             2019..2045
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             2019..2045
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2046..2099
                     /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(2100..2102,2103..2816)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2100..2102,2103..2813)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2142..2163)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2403..2422)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2750..2771
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        2827..3038
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             3060..3656
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3060..3079)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3556..3576
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3666..3722
                     /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             join(3723..4340,4341..4346,4347..4382,4383..4385,4386..4421,
                     4422..4424,4425..4460,4461..4469)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    4490..5078
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4543..4563)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4961..4972)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5150..5383
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5409..5426)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5415..5639
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5685..6113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5772..5791)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5982..6003
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6122..6142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6127..6456
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6307..6442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6369..6388
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        6504..6551
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             6570..6944
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    7074..7195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7111..7130)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7165..7184
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7244..7260)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7244..7260)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7257..7279)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7268..7284
                     /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)."
     promoter        complement(join(7292..7298,7299..7316,7317..7322))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7337..7358
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7475..7492)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7646..8234)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7726..7745)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8405..9196,9197..9265))
                     /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     9028..9047
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9266..9370)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9445..9464)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        9636..10015
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        10017..10215
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             10233..10413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    10460..10585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11078..11311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             11496..11540
                     /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    11838..11955
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        12006..12254
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     12006..12026
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     12177..12196
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
       61 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
      121 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
      181 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
      241 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
      301 accgagtcgg tgcttttttg aattcacttt ggccgcgaat cgatatgtcg agtttactcc
      361 ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga gaacgatgtc
      421 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag
      481 agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga gtttatccct
      541 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg
      601 aggtaggcgt gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat
      661 cgcctggaga ataccggttc tagagcgctg ccaccatgag ctcagagact ggcccagtgg
      721 ctgtggaccc cacattgaga cggcggatcg agccccatga gtttgaggta ttcttcgatc
      781 cgagagagct ccgcaaggag acctgcctgc tttacgaaat taattggggg ggccggcact
      841 ccatttggcg acatacatca cagaacacta acaagcacgt cgaagtcaac ttcatcgaga
      901 agttcacgac agaaagatat ttctgtccga acacaaggtg cagcattacc tggtttctca
      961 gctggagccc atgcggcgaa tgtagtaggg ccatcactga attcctgtca aggtatcccc
     1021 acgtcactct gtttatttac atcgcaaggc tgtaccacca cgctgacccc cgcaatcgac
     1081 aaggcctgcg ggatttgatc tcttcaggtg tgactatcca aattatgact gagcaggagt
     1141 caggatactg ctggagaaac tttgtgaatt atagcccgag taatgaagcc cactggccta
     1201 ggtatcccca tctgtgggta cgactgtacg ttcttgaact gtactgcatc atactgggcc
     1261 tgcctccttg tctcaacatt ctgagaagga agcagccaca gctgacattc tttaccatcg
     1321 ctcttcagtc ttgtcattac cagcgactgc ccccacacat tctctgggcc accgggttga
     1381 aaagcggcag cgagactccc gggacctcag agtccgccac accagaaccc cacccagtgt
     1441 tggagaagct tcggtccatt aataactata accccaaaga ttttgactgg aatctgaaac
     1501 atggccgggt tttcatcatt aagagctact ctgaggacga tattcaccgt tccattaagt
     1561 ataatatttg gtgcagcaca gagcatggta acaagagact ggatgctgct tatcgttcca
     1621 tgaacgggaa aggccccgtt tacttacttt tcagtgtcaa cggcagtgga cacttctgtg
     1681 gcgtggcaga aatgaaatct gctgtggact acaacacatg tgcaggtgtg tggtcccagg
     1741 acaaatggaa gggtcgtttt gatgtcaggt ggatttttgt gaaggacgtt cccaatagcc
     1801 aactgcgaca cattcgccta gagaacaacg agaataaacc agtgaccaac tctagggaca
     1861 ctcaggaagt gcctctggaa aaggctaagc aggtgttgaa aattatagcc agctacaagc
     1921 acaccacttc catttttgat gacttctcac actatgagaa acgccaagag gaagaagaaa
     1981 gtgttaaaaa ggaacgtcaa ggtcgtggga aactcgagta cccctacgac gtgcccgact
     2041 acgccgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
     2101 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2161 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2221 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2281 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2341 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2401 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2461 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2521 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2581 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2641 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2701 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2761 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaagcta
     2821 gcatccgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
     2881 attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
     2941 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
     3001 acgttctttt tcgcaacggg tttgccgcca gaacacagac tgcgatcgca atgtacagta
     3061 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     3121 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     3181 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     3241 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     3301 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     3361 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     3421 ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca
     3481 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     3541 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     3601 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggtt
     3661 ccggcgcaac aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac
     3721 cgatgtctag actggacaag agcaaagtca taaacggagc tctggaatta ctcaatggtg
     3781 tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt gagcagccta
     3841 ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca atcgagatgc
     3901 tggacaggca tcatacccac ttctgccccc tggaaggcga gtcatggcaa gactttctgc
     3961 ggaacaacgc caagtcatac cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc
     4021 atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc
     4081 tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc gtgggccact
     4141 ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag gaaagagaga
     4201 cacctaccac cgattctatg cccccacttc tgagacaagc aattgagctg ttcgaccggc
     4261 agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc ctggagaaac
     4321 agctaaagtg cgaaagcggc gggccgaccg acgcccttga cgattttgac ttagacatgc
     4381 tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac gctcttgacg
     4441 attttgacct tgacatgctc cccgggtaac tcgagcgcgt taagtcgaca atcaacctct
     4501 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     4561 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     4621 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4681 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4741 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4801 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4861 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     4921 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4981 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     5041 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     5101 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     5161 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     5221 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     5281 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     5341 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagggcccg tttaaacccg
     5401 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     5461 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     5521 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     5581 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     5641 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     5701 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     5761 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     5821 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     5881 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     5941 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     6001 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     6061 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     6121 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     6181 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     6241 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     6301 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     6361 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     6421 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     6481 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     6541 tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc
     6601 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     6661 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     6721 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     6781 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     6841 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     6901 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     6961 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     7021 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     7081 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     7141 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     7201 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     7261 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     7321 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     7381 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     7441 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     7501 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     7561 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     7621 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     7681 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     7741 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     7801 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     7861 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     7921 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     7981 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     8041 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     8101 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     8161 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     8221 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     8281 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     8341 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     8401 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     8461 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     8521 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     8581 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     8641 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     8701 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     8761 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     8821 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     8881 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     8941 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     9001 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     9061 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     9121 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     9181 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     9241 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     9301 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     9361 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     9421 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     9481 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac
     9541 aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc
     9601 gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta
     9661 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     9721 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     9781 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     9841 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     9901 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     9961 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
    10021 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
    10081 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
    10141 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
    10201 ggtctatata agcagcgcgt tttgcctgta ctgggtctct ctggttagac cagatctgag
    10261 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
    10321 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
    10381 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
    10441 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
    10501 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
    10561 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
    10621 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
    10681 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
    10741 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
    10801 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
    10861 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
    10921 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
    10981 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
    11041 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
    11101 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
    11161 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
    11221 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
    11281 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
    11341 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
    11401 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
    11461 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
    11521 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
    11581 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
    11641 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
    11701 ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag
    11761 acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg
    11821 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
    11881 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
    11941 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gttaattaag
    12001 gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
    12061 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
    12121 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
    12181 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
    12241 aaggacgaaa caccggagac ggttgtaaat gagcacacaa aatacacatg ctaaaatatt
    12301 atattctatg acctttataa aatcaaccaa aatcttcttt ttaataactt tagtatcaat
    12361 aattagaatt tttatgttcc tttttgcaaa cttttaataa aaatgagcaa aataaaaaaa
    12421 cgctagtttt agtaactcgc gttgttttct tcacctttaa taatagctac tccaccactt
    12481 gttcctaagc ggtcagctcc tgcttcaatc attttttgag catcttcaaa tgttctaact
    12541 ccaccagctg ctttaactaa agcattgtct ttaacaactg acttcattag tttaacatct
    12601 tcaaatgttg cacctgattt tgaaaatcct gttgatgttt taacaaattc taatccagct
    12661 tcaacagcta tttcacaagc tttcatgatt tcttcttttg ttaataaaca attttccata
    12721 atacatttaa caacatgtga tccagctgct ttttttacag ctttcatgtc ttctaaaact
    12781 aattcataat ttttgtcttt taatgcacca atatttaata ccatatcaat ttctgttgca
    12841 ccatctttaa ttgcttcaga aacttcgaat gcttttgtag ctgttgtgca tgcacctaga
    12901 ggaaaaccta caacatttgt tattcctaca tttgtgcctt ttaataattc tttacaatag
    12961 cttgttcaat atgaattaac acaaactgtt gcaaaatcaa attcaattgc ttcatcacat
    13021 aattgtttaa tttcagcttt cgtagcatct tgttttaata atgtgtgatc tatatatttg
    13081 tttagtttca ttttttctcc tatatattca tttttaattt taattcttta ataatttcgt
    13141 ctactttaac tttagcgttt tgaacagatt caccaacacc tataaaataa atttttagtt
    13201 taggttcagt tccacttggg cgaacagcaa atcatgactt atcttctaaa taaaatttta
    13261 gtaagtcttg tcctggcata ttatacattc catcgatgta gtcttcaaca ttaacaactt
    13321 taagtccagc aatttgagtt aagggtgttg ctctcaatga tttcattaat ggttcaattt
    13381 ttaatttctt ttcttctggt ttaaaattca agtttaaagt gaaagtgtaa tatgcaccca
    13441 tttctttaaa taaatcttct aaatagtcta ctaatgtttt attttgtttt ttataaaatc
    13501 aagcagcctc tgctattaat atagaagctt gtattccatc tttatctcta gctgagtcat
    13561 caattacata tccataactt tcttcataag caaaaacaaa atttaatccg ttatcttctt
    13621 ctttagcaat ttctctaccc attcatttaa atccagttaa agtttttaca atattaactc
    13681 catatttttc atgagcgatt ctatcaccca aatcacttgt tacaaaactt gaatatagag
    13741 ccggattttt tggaatgcta tttaagcgtt ttagatttga taattttcaa tcaattaaaa
    13801 ttggtcctgt ttgatttcca tctaatctta caaaatgacc atcatgtttt attgccattc
    13861 caaatctgtc agcatctggg tcattcataa taataata
//
