LOCUS       sequence-461778-       10194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bxb1-GA donor plasmid with convergent syntax for all-in-one 
            doxycycline-inducible mScarlet expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10194)
  AUTHORS   Blanch-Asensio A, Ploessl DS, Wang NB, Mummery CL, Galloway KE, 
            Davis RP
  TITLE     STRAIGHT-IN Dual: a platform for dual, single-copy integrations of 
            DNA payloads and gene circuits into human induced pluripotent stem 
            cells.
  JOURNAL   bioRxiv [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637.
  PUBMED    39464154
REFERENCE   2  (bases 1 to 10194)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2024 Oct 17:2024.10.17.616637. doi: 
            10.1101/2024.10.17.616637."
COMMENT     Sequence Label: Bxb1-GA_AIO-TetOn_mScarlet_Convergent
FEATURES             Location/Qualifiers
     source          1..10194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        275..471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        558..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1684..1704
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1744..1753
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1756..1793
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     protein_bind    1808..1855
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     enhancer        1897..2276
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3444..3466
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3528..3547
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3579..4322
                     /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             4323..4376
                     /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             4377..5084
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
     CDS             5109..5129
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5133..5153
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5157..5177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5242..5259)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5248..5455
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    5511..6099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             5617..5628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     6026..6046
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6103..6798)
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(6378..6397)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6647..6665
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      6795..6804
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(6883..6907)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    6952..6970
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6988..7006
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7024..7042
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7060..7078
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7096..7114
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7132..7150
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7168..7186
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7252..7285
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     primer_bind     complement(7293..7309)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7323..7339)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7332..7354)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7552..8007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7639..7658)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7849..7870
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8024..8043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8143..8165
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8203..8221)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8289..8393
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8394..8462,8463..9254)
                     /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(8612..8631)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9425..10013
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9914..9933
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10167..10184
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttcgccacca tggccggctt gtcgacgacg gcggactccg tcgtcaggat catccggatc
     1801 cggatccgaa gttcctattc cgaagttcct attctctaga aagtatagga acttcatgat
     1861 attcggcaag caggcatcga ctagttaatt aagctcgaca ttgattattg actagttatt
     1921 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     1981 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     2041 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     2101 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     2161 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccat tacatgccct
     2221 taggggcctt tcctacttgg cagtacatct acgtattagt catcgctatt cccatggtcg
     2281 aggtgagccc cacgttctgc ttcactctcc ccatcccccc cccccccccc cccccaattt
     2341 tttttttttt tttttttaat ttgtttgggg cggggggggg gggggggggg gggggcgcgc
     2401 gccgggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc
     2461 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
     2521 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt
     2581 gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc
     2641 acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg
     2701 acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg agggcccttt
     2761 gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg
     2821 cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct
     2881 ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg
     2941 cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg
     3001 cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg agcgcggccc
     3061 ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg
     3121 gtggcggcag gtgggggtgc cgggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     3181 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     3241 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     3301 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     3361 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     3421 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     3481 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     3541 tattgtgctg tctcatcatt ttggcaaaga atgtcgacat gagccggctg gacaagagca
     3601 aagtgatcaa cagcgccctg gaactgctga acggcgtggg catcgagggc ctgaccaccc
     3661 ggaagctggc ccagaaactg ggcgtggaac agcccaccct gtactggcac gtgaagaaca
     3721 agcgggccct gctggacgcc ctgcccatcg agatgctgga ccggcaccac acccacagct
     3781 gccctctgga aggcgagagc tggcaggact tcctgcggaa caacgccaag agctacagat
     3841 gcgccctgct gagccaccgg gacggcgcca aagtgcacct gggcaccaga cccaccgaga
     3901 agcagtacga gacactggaa aaccagctgg ccttcctgtg ccagcagggc ttcagcctgg
     3961 aaaacgccct gtacgccctg agcgccgtgg gccacttcac cctgggctgc gtgctggaag
     4021 aacaggaaca ccaggtcgcc aaagaggaaa gagagacacc caccaccgac agcatgcccc
     4081 ccctgctgaa gcaggccatc gagctgttcg acagacaggg cgccgagccc gccttcctgt
     4141 tcggcctgga actgatcatc tgcggcctcg agaagcagct gaagtgcgag agcggcggac
     4201 ccaccgacgc cctggacgac ttcgacctgg acatgctgcc cgccgatgcc ctggatgatt
     4261 ttgatctgga tatgctccct gctgacgctc tcgatgactt cgatctcgat atgctgccag
     4321 gcgagggcag gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc ggccccgtgt
     4381 ctaagggcga agagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
     4441 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
     4501 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
     4561 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
     4621 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
     4681 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
     4741 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     4801 gctgggaggc cttcaccgag acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     4861 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacgccaag accacatata
     4921 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     4981 tggaaagaat caaggaggcc aacaacgaga cctacgtcga gcagcacgag gtggcagtgg
     5041 ccagatactg cgacctccct agcaaactgg ggcacaagct taattccgga ctcagatctc
     5101 gagctgatcc aaaaaagaag agaaaggtag atccaaaaaa gaagagaaag gtagatccaa
     5161 aaaagaagag aaaggtagga tccaccggat ctagataact gatcataatc agccatacca
     5221 catttgaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5281 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5341 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     5401 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     5461 tgggctctat cccgcatccc cagcactagc gcggggaggc ggcccaaagg gcggggaggc
     5521 ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag
     5581 agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag ggacgtagca
     5641 gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccatg gaaaggacgt
     5701 cagcttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc ctgtccagca
     5761 gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga aagcgaaagt
     5821 cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg cgtcagcaaa
     5881 cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat aaagagacag
     5941 caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa tagcatgata
     6001 caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt taagaatacc
     6061 agtcaatctt tcacaaattt tgtaatccag aggttgattt tacttgtaca gctcgtccat
     6121 gccgccggtg gagtggcggc cctcggagcg ttcgtactgt tccaccacgg tgtagtcctc
     6181 gttgtgggag gtgatgtcca acttgcggtc gacgttgtag gcgccgggca tctgcacggg
     6241 cttcttggcc ttgtaggtgg tcttgaagtc cgccaggtag cggccgccgt ccttcaggcg
     6301 cagggccatc ttaatgtcgc ccttcagcac gccgtcctcg gggtacaacc gctcggtgga
     6361 cgcttcccag cccattgtct tcttctgcat tacggggccg tcaggaggga agttggtgcc
     6421 gcggagcttc accttgtaga tcagggtgcc gtcctccagg gaggtgtcct gggtcacggt
     6481 cacggcgccg ccgtcctcga agttcatcac gcgctcccac ttgaagccct cggggaagga
     6541 ctgcttatag tagtcgggga tgtcggcggg gtgcttggtg aaggccctgg agccgtacat
     6601 gaactgaggg gacaggatgt cccaggagaa gggcaggggg ccacccttgg tcaccttcag
     6661 cttggcggtc tgggtgccct cgtaggggcg gccctcgccc tcgccctcga tctcgaactc
     6721 gtggccgttc atggagccct ccatgtgcac cttgaaccgc atgaactcct tgatcactgc
     6781 ctcgcccttg ctcaccatgg tggcgaattc ttaagtttac gagggtagga agtggtacgg
     6841 aaagttggta taagacaaaa gtgttgtgga attgctccag gcgatctgac ggttcactaa
     6901 acgagctctg cttttatagg cgcccaccgt acacgcctaa agcttatacg ttctctatca
     6961 ctgataggga gtaaactgga tatacgttct ctatcactga tagggagtaa actgtagata
     7021 cgttctctat cactgatagg gagtaaactg gtcatacgtt ctctatcact gatagggagt
     7081 aaactcctta tacgttctct atcactgata gggagtaaag tctgcatacg ttctctatca
     7141 ctgataggga gtaaactctt catacgttct ctatcactga tagggagtaa accttaatta
     7201 actagtcgcc gctgcaagaa ctcttcctca cggatccgaa gttcctattc cgaagttcct
     7261 attcttcaaa tagtatagga acttcagcgc tcactggccg tcgttttaca aagggcgaat
     7321 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     7381 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     7441 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     7501 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     7561 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     7621 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     7681 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     7741 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     7801 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     7861 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     7921 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     7981 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     8041 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     8101 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     8161 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     8221 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     8281 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     8341 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     8401 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     8461 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     8521 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     8581 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     8641 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     8701 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     8761 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     8821 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     8881 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     8941 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     9001 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     9061 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     9121 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     9181 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     9241 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     9301 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     9361 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     9421 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     9481 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     9541 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     9601 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     9661 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     9721 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     9781 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     9841 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     9901 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     9961 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
    10021 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
    10081 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
    10141 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaag
//
