LOCUS       sequence-430995-       12318 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Homologous recombination donor to integrate and expression cassette 
            for EGFP-GABRAPL1 in the AAVS1 locus of human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12318)
  AUTHORS   Broadbent DG, Barnaba C, Perez GI, Schmidt JC
  TITLE     Quantitative analysis of autophagy reveals the role of ATG9 and ATG2
            in autophagosome formation.
  JOURNAL   J Cell Biol. 2023 Jul 3;222(7):e202210078. doi: 
            10.1083/jcb.202210078. Epub 2023 Apr 28.
  PUBMED    37115157
REFERENCE   2  (bases 1 to 12318)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2023-07-3"; volume: "222(7):e202210078. doi"; pages: 
            " 10.1083/jcb.202210078. Epub 2023 Apr 28"
COMMENT     Sequence Label: AAVS1 EGFP-GABARAPL1 HRD
FEATURES             Location/Qualifiers
     source          1..12318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(3..21)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(42..58)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    66..82
                     /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(90..96,97..114,115..120))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    135..156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(273..290)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(444..1032)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(524..543)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             join(1156..1224,1225..2016)
                     /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(1374..1393)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2377..2396)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2991..3010
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(3413..3715)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(3515..3534)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     3838..3860
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3852..3869
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3853..3869
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3876..3895
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3876..3894
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3929..4765
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    4839..4860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4875..4880,4881..4898,4899..4905)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4913..4929
                     /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     4937..4953
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5296..5315
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    5314..5369
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     5415..5434
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(5525..6271)
                     /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"
     primer_bind     complement(6309..6328)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          6336..7352
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(6390..6412)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(7353..7628)
                     /label=chicken beta-actin promoter
     enhancer        7630..7933
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8017..8395
                     /label=TRE3G promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G"
     protein_bind    8025..8043
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8061..8079
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8097..8115
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8133..8151
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8169..8187
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8205..8223
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    8241..8259
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     8304..8328
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(8402..8404,8405..8407,8408..9118)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(8402..8404,8405..8407,8408..9118)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8447..8468)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(8708..8727)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9055..9076
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             9131..9151
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     intron          9700..9765
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             9895..9915
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    10340..10461
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10377..10396)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10431..10450
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    10499..10723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     10712..10729
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(10761..11360)
                     /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(10844..10864)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     11341..11360
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(11370..11423)
                     /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"
     misc_feature    11447..11472
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    11479..12282
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 tccctttagt gagggttaat tctgagcttg gcgtaatcat ggtcatagct gtttcctgtg
       61 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
      121 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
      181 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
      241 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
      301 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
      361 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
      421 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
      481 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      541 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
      601 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
      661 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
      721 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
      781 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
      841 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
      901 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
      961 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1021 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1081 aactcacgtt aagggatttt ggtcatgaga caataaccct gataaatgct tcaataatat
     1141 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1201 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1261 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1321 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1381 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     1441 tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac ggatggcatg
     1501 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1561 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1621 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1681 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1741 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1801 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     1861 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     1921 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     1981 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2041 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2101 tttgataatc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     2161 gttccgcgca catttccccg aaaagtgcca cctgatgcgg tgtgaaatac cgcacagatg
     2221 cgtaaggaga aaataccgca tcaggaaatt gtaagcgtta ataattcaga agaactcgtc
     2281 aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag
     2341 gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat
     2401 gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc
     2461 attttccacc atgatattcg gcaagcaggc atcgccatgc atgggtcacg acgagatcct
     2521 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     2581 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     2641 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     2701 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     2761 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     2821 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     2881 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     2941 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     3001 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     3061 tcatgcgaaa cgatcctcat cctgtctctt gatcagagct tgatcccctg cgccatcaga
     3121 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
     3181 gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc
     3241 gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc
     3301 agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct
     3361 acgtgaaaag gatctaggtg aagatccttt ttgataatct catgcctgac atttatattc
     3421 cccagaacat caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca
     3481 cttcttcccc gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc
     3541 tttcatcccc gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac
     3601 gtgcactggc cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta
     3661 catccacaaa cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta
     3721 cgtataggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     3781 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     3841 agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat
     3901 tgaatttagc ggccgcgaat tcgcccttag agcagagcca ggaacccctg tagggaaggg
     3961 gcaggagagc caggggcatg agatggtgga cgaggaaggg ggacagggaa gcctgagcgc
     4021 ctctcctggg cttgccaagg actcaaaccc agaagcccag agcagggcct tagggaagcg
     4081 ggaccctgct ctgggcggag gaatatgtcc cagatagcac tggggactct ttaaggaaag
     4141 aaggatggag aaagagaaag ggagtagagg cggccacgac ctggtgaaca cctaggacgc
     4201 accattctca caaagggagt tttccacacg gacacccccc tcctcaccac agccctgcca
     4261 ggacggggct ggctactggc cttatctcac aggtaaaact gacgcacgga ggaacaatat
     4321 aaattgggga ctagaaaggt gaagagccaa agttagaact caggaccaac ttattctgat
     4381 tttgtttttc caaactgctt ctcctcttgg gaagtgtaag gaagctgcag caccaggatc
     4441 agtgaaacgc accagacagc cgcgtcagag cagctcaggt tctgggagag ggtagcgcag
     4501 ggtggccact gagaaccggg caggtcacgc atccccccct tccctcccac cccctgccaa
     4561 gctctccctc ccaggatcct ctctggctcc atcgtaagca aaccttagag gttctggcaa
     4621 ggagagagat ggctccagga aatgggggtg tgtcaccaga taaggaatct gcctaacagg
     4681 aggtgggggt tagacccaat atcaggagac taggaaggag gaggcctaag gatggggctt
     4741 ttctgtcacc aatcctgtcc ctagtaaagc ttagtactgt cgatccgctg gcacgacagg
     4801 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     4861 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     4921 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct tgggctgcag
     4981 gtcgagggat ctccataaga gaagagggac agctatgact gggagtagtc aggagaggag
     5041 gaaaaatctg gctagtaaaa catgtaagga aaattttagg gatgttaaag aaaaaaataa
     5101 cacaaaacaa aatataaaaa aaatctaacc tcaagtcaag gcttttctat ggaataagga
     5161 atggacagca gggggctgtt tcatatactg atgacctctt tatagccaac ctttgttcat
     5221 ggcagccagc atatgggcat atgttgccaa actctaaacc aaatactcat tctgatgttt
     5281 taaatgattt gccctcccat atgtccttcc gagtgagaga cacaaaaaat tccaacacac
     5341 tattgcaatg aaaataaatt tcctttatta gccagaagtc agatgctcaa ggggcttcat
     5401 gatgtcccca taatttttgg cagagggaaa aagatctcag tggtatttgt gagccagggc
     5461 attggccaca ccagccacca ccttctgata ggcagcctgc acctgaggag tgaattcacg
     5521 cgccttaccc ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat
     5581 caaggtcaaa gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg
     5641 cgtcggtcgg cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt
     5701 ccaggccgaa aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtt
     5761 tcagaagtgg gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat
     5821 gctcctgttc ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca
     5881 gtgcgttctc cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt
     5941 cgtactgttt ctctgttggg cgggtgccga gatgcacttt agccccgtcg cgatgtgaga
     6001 ggagagcaca gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt
     6061 ccagggggca ggagtgggta tgatgcctgt ccagcatctc gattggcagg gcatcgagca
     6121 gggcccgctt gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag
     6181 cgagtttcct tgtcgtcagg ccttcgatac cgactccatt gagtaattcc agagcagagt
     6241 ttatgacttt gctcttgtcc agtctagaca tggtgctcga cgaattcttt gccaaaatga
     6301 tgagacagca caataaccag cacgttgccc aggagctgta ggaaaaagaa gaaggcatga
     6361 acatggttag cagaggctct agagccgccg gtcacacgcc agaagccgaa ccccgccctg
     6421 ccccgtcccc cccgaaggca gccgtccccc cgcggacagc cccgaggctg gagagggaga
     6481 aggggacggc ggcgcggcga cgcacgaagg ccctccccgc ccatttcctt cctgccggcg
     6541 ccgcaccgct tcgccccgcg cccgctagag ggggtgcggc ggcgcctccc agatttcggc
     6601 tccgcacaga tttgggacaa aggaagtccc tgcgccctct cgcacgatta ccataaaagg
     6661 caatggctgc ggctcgccgc gcctcgacag ccgccggcgc tccgggggcc gccgcgcccc
     6721 tcccccgagc cctccccggc ccgaggcggc cccgccccgc ccggcacccc cacctgccgc
     6781 caccccccgc ccggcacggc gagccccgcg ccacgccccg tacggagccc cgcacccgaa
     6841 gccgggccgt gctcagcaac tcggggaggg gggtgcaggg ggggttgcag cccgaccgac
     6901 gcgcccacac cccctgctca cccccccacg cacacacccc gcacgcagcc tttgttcccc
     6961 tcgcagcccc ccccgcaccg cggggcaccg cccccggccg cgctcccctc gcgcacactg
     7021 cggagcgcac aaagccccgc gccgcgcccg cagcgctcac agccgccggg cagcgcggag
     7081 ccgcacgcgg cgctccccac gcacacacac acgcacgcac cccccgagcc gctccccccg
     7141 cacaaagggc cctcccggag cccctcaagg ctttcacgca gccacagaaa agaaacaagc
     7201 cgtcattaaa ccaagcgcta attacagccc ggaggagaag ggccgtcccg cccgctcacc
     7261 tgtgggagta acgcggtcag tcagagccgg ggcgggcggc gcgaggcggc ggcggagcgg
     7321 ggcacggggc gaaggcagcg cgcagcgact cccgcccgcc gcgcgcttcg ctttttatag
     7381 ggccgccgcc gccgccgcct cgccataaaa ggaaactttc ggagcgcgcc gctctgattg
     7441 gctgccgccg cacctctccg cctcgccccg ccccgcccct cgccccgccc cgccccgcct
     7501 ggcgcgcgcc cccccccccc ccgcccccat cgctgcacaa aataattaaa aaataaataa
     7561 atacaaaatt gggggtgggg agggggggga gatggggaga gtgaagcaga acgtggggct
     7621 cacctcgacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     7681 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     7741 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     7801 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     7861 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     7921 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     7981 ctattaataa ctagttttcg tcttcaagaa ttcctcgagt ttactcccta tcagtgatag
     8041 agaacgtatg aagagtttac tccctatcag tgatagagaa cgtatgcaga ctttactccc
     8101 tatcagtgat agagaacgta taaggagttt actccctatc agtgatagag aacgtatgac
     8161 cagtttactc cctatcagtg atagagaacg tatctacagt ttactcccta tcagtgatag
     8221 agaacgtata tccagtttac tccctatcag tgatagagaa cgtataagct ttaggcgtgt
     8281 acggtgggcg cctataaaag cagagctcgt ttagtgaacc gtcagatcgc ctggagcaat
     8341 tccacaacac ttttgtctta taccaacttt ccgtaccact tcctaccctc gtaaagtcga
     8401 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     8461 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     8521 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     8581 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     8641 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     8701 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     8761 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     8821 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     8881 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     8941 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     9001 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     9061 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     9121 gccaaccact gaggatctgt actttcagag cgataacgcg atcgcttccg gtaccggcgg
     9181 ccgcaagttc cagtacaagg aggaccatcc ctttgagtat cggaaaaagg aaggagaaaa
     9241 gatccggaag aaatatccgg acagggtccc cgtgattgta gagaaggctc caaaagccag
     9301 ggtgcctgat ctggacaaga ggaagtacct agtgccctct gaccttactg ttggccagtt
     9361 ctacttctta atccggaaga gaatccacct gagacctgag gacgccttat tcttctttgt
     9421 caacaacacc atccctccca ccagtgctac catgggccaa ctgtatgagg acaatcatga
     9481 ggaagactat tttctgtatg tggcctacag tgatgagagt gtctatggga aatctagaat
     9541 gtgatcatat ggctagcctg cagggatcca atgtaactgt attcagcgat gacgaaattc
     9601 ttagctattg taatactcta gaggatcttt gtgaaggaac cttacttctg tggtgtgaca
     9661 taattggaca aactacctac agagatttaa agctctaagg taaatataaa atttttaagt
     9721 gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattcc aacctatgga
     9781 actgatgaat gggagcagtg gtggaatgcc tttaatgagg aaaacctgtt ttgctcagaa
     9841 gaaatgccat ctagtgatga tgaggctact gctgactctc aacattctac tcctccaaaa
     9901 aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag ttttttgagt
     9961 catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac aaaggaaaaa
    10021 gctgcactgc tatacaagaa aattatggaa aaatattctg taacctttat aagtaggcat
    10081 aacagttata atcataacat actgtttttt cttactccac acaggcatag agtgtctgct
    10141 attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa aggggttaat
    10201 aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt
    10261 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
    10321 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
    10381 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
    10441 caaactcatc aatgtatctt atcatgtctg cggcctagtc aataatcaat gtcgagaccc
    10501 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
    10561 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
    10621 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
    10681 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcgg
    10741 gtttaaacgg gccctctaga tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
    10801 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
    10861 ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
    10921 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg
    10981 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
    11041 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
    11101 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
    11161 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
    11221 gtgacccgct cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg gtagtcggcg
    11281 aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc gaggcgcacc
    11341 gtgggcttgt actcggtcat ctcgagccta gggccgggat tctcctccac gtcaccgcat
    11401 gttagaagac ttcctctgcc ctctccgctg ccagatctct cgaggccctg tgggaggaag
    11461 agaagaggtc agaagcttgc cccactgtgg ggtggagggg acagataaaa gtacccagaa
    11521 ccagagccac attaaccggc cctgggaata taaggtggtc ccagctcggg gacacaggat
    11581 ccctggaggc agcaaacatg ctgtcctgaa gtggacatag gggcccgggt tggaggaaga
    11641 agactagctg agctctcgga cccctggaag atgccatgac agggggctgg aagagctagc
    11701 acagactaga gaggtaaggg gggtagggga gctgcccaaa tgaaaggagt gagaggtgac
    11761 ccgaatccac aggagaacgg ggtgtccagg caaagaaagc aagaggatgg agaggtggct
    11821 aaagccaggg agacggggta ctttggggtt gtccagaaaa acggtgatga tgcaggccta
    11881 caagaagggg aggcgggacg caagggagac atccgtcgga gaaggccatc ctaagaaacg
    11941 agagatggca caggccccag aaggagaagg aaaagggaac ccagcgagtg aagacggcat
    12001 ggggttgggt gagggaggag agatgcccgg agaggaccca gacacgggga ggatccgctc
    12061 agaggacatc acgtggtgca gcgccgagaa ggaagtgctc cggaaagagc atccttgggc
    12121 agcaacacag cagagagcaa ggggaagagg gagtggagga agacggaacc tgaaggaggc
    12181 ggcagggaag gatctgggcc agccgtagag gtgacccagg ccacaagctg cagacagaaa
    12241 gcggcacagg cccaggggag agaatgctgg tcagagaaag caaagggcga attcgtttaa
    12301 acctgcagga ctagctag
//
