Supplemental Table S1: Clinical details of patients with mutations in non-SMC subunits

Condensin I subunits Condensin II subunits

Gene NCAPD2 NCAPH NCAPD3 NCAPD3

P1 P3 P2 P4

Mutation details

c.[382+14A>G] Nucleotide c.4120+2T>C c.728C>T c.3458T>G +[1783_1784delG]

p.Ser129Metfs*1 p.Asp1374Glyfs*29 p.Pro243Leu p.Glu1153Ala p.Val595Serfs*34

Country of Origin

India Portugal USA UK

Pre-natal growth parameters

Gestn 37 N/A 37 39 /weeks

Weight -4.88 N/A -1.85 1.14

/s.d.(kg) (1) ‘small at birth’ (2.15) (3.88)

OFC N/A N/A N/A `Normal range` /s.d.(cm)

Length -4 N/A N/A `Normal range` /s.d.(cm) (40.6)

Post-natal growth parameters and clinical features

Age of exam 3y 42y 6y5m 6y11m

OFC -11.9 -4.2 -5.4 -2.7

/s.d.(cm) (34.8) (51.1) (45) (49.3)

Height -5.8 -1.4 -5.7 -1.3

/s.d.(cm) (74) (168.5) (90) (114.5)

Weight -7.25 -9.47 -1.95 N/A /s.d.(kg) (7) (9.07) (18)

Severe intellectual Moderate intellectual Development Normal Moderate delay disability, no speech disability

OFC, occupitofrontal circumference. s.d. standard deviations from the population mean.

Supplemental Figure S1: Condensin patients have severe disproportionate microcephaly

(A) Growth parameters plotted as standard deviations (z-score) from the population mean for age and sex. Red diamond, P1; Purple square, P2; Blue circle, P3; Green triangle, P4. OFC, occipitofrontal circumference. (B) Photographs of condensin patients. Aside from microcephaly, a consistent facial appearance is not evident in individuals with mutations in NCAPD3, NCAPD2 and NCAPH. Images of patient P3 from Figure 1A, included as a comparison.

Supplemental Figure S2: Consequences of condensin missense mutations on protein levels

(A) NCAPD2 protein levels are significantly reduced in P1 patient fibroblasts. Quantification of NCAPD2 protein levels in P1 primary fibroblasts. Chemiluminescence from western blots quantified using ImageQuant. Error bars, s.e.m, n=3 experiments; values normalized to actin signal; p value one-sample t test ** p≤0.01 (B) Condensin II complex , CAPG2 and CAPH2, are expressed at normal levels in P2 and P4 patient fibroblasts. Immunoblot demonstrates residual NCAPD3 protein is detectable in P2 at higher exposures whereas CAPG2 and CAPH2 protein levels are unaffected in P2 and P4 cells. Lower panel; loading control, blot probed with anti-actin antibody. (C) Schematic of NCAPH protein domain structure with location of patient mutation. (D) The Pro243Leu missense mutation identified in NCAPH does not affect protein levels in patient fibroblasts. NCAPH immunoblot demonstrating that protein levels are unaffected in P3 fibroblasts with p.Pro243Leu mutation. Left two lanes; RNAi of NCAPD2 in RPE1 cells demonstrating specificity of NCAPH antibody. Remaining lanes; P3 and control primary fibroblasts. Lower panel, loading control, blot probed with anti-Actin antibody.

Supplemental Figure S3: Brain and body weight is significantly reduced in Ncaph2I15N/I15N mice at 8 weeks

(A) Brain weights and (B) body weights at 8 weeks of age, expressed as a z-score, plotted relative to Ncaph2I15N/+ mice of the same age. z-score defined as the standard deviations from the mean for sex- matched Ncaph2I15N/+ mice at 8 weeks. N>17 mice per genotype, error bars, s.e.m. P value, two- tailed t-test, **≤0.01, ***≤0.001, versus Ncaph2I15N/+ mice.

Supplemental Figure S4. 3D spindle orientation is not altered in Ncaph2I15N/I15N mice

(A,B) Measurements of cleavage plane in 3D in apical progenitor cells. (A) Imaris 3D rendered image depicting points used to define the mitotic spindle and ventricular surface. DNA, Beige; centrosomes, white circles (defined by aurora A staining); plane of ventricular surface, dashed lines (defined by reference points (blue circles)). (B) Schematic of the angle calculation between the spindle line vector and the reference plane for ventricular surface. Green circles, centrosomes; red circle, point where spindle line vector intersects the reference plane. (C) Quantification of the cleavage plane orientation, categorized into 0-30°,30-60°, 60-90° bins. For each experiment, percentage of in each bin were calculated from the data in Figure 3G. N=3 exp, error bars, s.e.m. Graph ns= non-significant with an unpaired t- test.

Supplemental Figure S5: segregation in Ncaph2I15N/I15N embryonic fibroblasts and patient primary fibroblast lines

(A) Chromosome segregation is impaired in Ncaph2I15N/I15N embryonic fibroblasts. Quantification of chromosome segregation defects ( bridges and lagging ) observed in mid and late anaphase cells from Ncaph2I15N/I15N embryonic fibroblasts and Ncaph2I15N/+ litter-mate controls (expt=3, n>90 anaphases). Error bars, s.e.m. P value, two-tailed t-test, **≤0.01, versus Ncaph2I15N/+ #2 line. (B) A large proportion of persisting UFBs in condensin II deficient cells originate from centromeres. Top panel, UFB (detected by presence of PICH, red) flanked by centromeres (HEC1, green). Scale bar =10µm. Bottom panel, quantification of UFBs emanating from centromeric loci during anaphase in C2, P1 and P2. UFBs with HEC1 signal at one or both ends was scored as positive. Expt=3, n>94 mid/late anaphases. P value, x2 test ****≤0.0001. (C) Increased numbers of chromatin bridges in live imaging of condensin II patient fibroblasts. Quantification of the chromosome segregation defects observed in control and patient fibroblast cells during live imaging. Increased numbers of anaphase cells with chromatin bridges are seen in NCAPD3 P2 patient cell fibroblasts compared to C1 and C2 fibroblasts (n>148 cells from >3 exp). P value, X2 test, *≤0.01 versus C1.

Supplemental Table S2 A) Primer sequences for PCR amplification and sequencing of condensin exons Name Forward sequence Reverse sequence

NCAPD3 1 GATTGGTCCACAGGAACG ACGCATAGGACCCTCGC 2 TCTCCTCTTGAACACACCTGG CCTGTGAACTGTACACCAAGAAAG 3 GGAGCCAGGCTACTCTATTCC TCTCTGCCATGGGTTAGCAC 4 CATGAAAAGCCTGTGTTTTGC TGCCTATAATCATGATGCCTG 5-6 CCATGGGACAAAGTGTTTGG TGGTATTTCCATTTCTGCTGG 7 CATTTAAACATGGCTTGTAGGAC TATGGTTCATGTGCCTTCG 8 TCTGAGACCCTTGTGTTCTCC CTTAGTGCAGGGCCCAGAG 9 CCACTCTTATAGCGTGAGGTAGAG AACAATGCACACTCATTCCC 10 AATATGTTTCAGCCATCCTGC CCAAAGAACCCTCCTCATTC 11 TGAGGAGGGTTCTTTGGTTC AAAGAACGATTCCCTTGCAG 12-13 TCTTGATTTTGATTCCTCCG ACGGTTGTCTGAAATCCAGC 14 TGGTGAAATGTTAGCTGCTG ACAACAGGATCAGGGTCAAC 15 CAGTTTATCTTTGTGAGTTGATGC GCAGCAGGTAATTATTGTGTGG 16 GGTCTATTTGCAGTTGAAGGTG CATACACTAAGTAATCTGGAAAGCT 17 CCTGGAGCAGCAGAAGTTG TCACTTCCTTAAGTCTTGGGC 18-19 TTGGCAGCAGTGTTTAACTTG CAAAACACATACAGTAACATCCTAAGC 20 AACATGGTGTTTCATGGGTAG AGAACCTCACGGATAAAATGG 21-22 GGGATAAAGGAAATACTCCATTGAC CAGCCCCTGACACAGTCC 23 AAATTTAGGAATGGAATTAGGATTTAG TCCCTTAAACCAGAAGCAATG 24 CTGGGAACAGTGAGGCTTG CCAGCATACATCAGAAATGAGG 25 TTGGTGTATTCAGAGACTGTTGG AGCCTAACCTCCCCTGATTC 26 CAGTGTTAAATCCCTTACACCC CTGGGCCATTATCAACACAG 27 AATTACACCTCCTCTCTGGGC CTTAGGGGAGCAGCACACTC 28 TGTGGTAGGGTGGCTCTACTC AATCATTCACGGATTGCCTG 29 TTGTTACACAGGCGAAGGTG GATGACGGAGATCTTGAGAGG 30 TGATTTGATCATTACGTTGTCC ACAATGACTTTCCTGCCCAC 31 GTGCAGCACTGTCACCTCTG CACCGCTTTGAAAGGAATAAG 32 TGTGGTTGTTTTGCTCTTCG ACACATCACGAATGCAGGAG 33 CCCACGGACAACTAGAACAG CATTCAGCTTTCCCAGAACC 34-35 CCTTCTGGGCTGATAGATGC GGACACGAGACTGCTTCCTC

NCAPD2 2 CAGGTCTTGAATATAGACCCTGAC CCATGTGAGATGGTCCTTTG 3-4 TTTTCTGCCATGGATAGAATTTAC CATGTGGCTGACAAAATGTAGG 5 TGCTGGGTGTTTTGAAGTTG TGGATCATTGGATTCCCTTC 6 TGGTACTAGATTGTTTGGGCAC TCAACAGAGCCTCACTCTCAC 7-8 TAGAATGAATGAGGCAGGGC TCTTTATTCCCTCCCCAAGG 9 CCTTCCCTTACTGGGAACTG TTGTCAAAGCTACTTTCTTGTTCTG 10 AGTTATGGCCAAGCACAAGC CACTGGATTCCTCTTTACACTGG 11-12 GAACAAAATGGATGATGTGGG TTTCTGACTGCAAGGAGAAGC 13 AGACAGGCTTCTCCTTGCAG CACCTGGTGGCAGCATATC 14 TTACAAACGGCTGCAGTGAC AGGCCAATGATGGCATAAAG 15 GTTGGATTCCCCTGTATCCC GGAAGGCCCCATCTCTATTG 16 TTCAGAAAGGTTCTGTCGTTAGG GAAGTCCTGTGAGAAGCTGACC 17 TCCAGATCCATAGGCAGTCC GATCACGAGGTCAAGGGTTC 18 GCAATATCATCTTCAGAAAAGCAG CCTCTCAGAACCTTCCTTTGC 19-20 CCCCGATGAGTCCAATCC CAGAGGAGGAGGGAGAAGTTG 21 CACAAAAGGTGAGCTCTCAGG CCTGCAGGGAGAACAGAAG 22 TGAATAGCACGTGAGCAAGG AGCAGAGGAGCAGAGATTGC 23-24 CATGCAAAACAAAGTATGGGC AAGCCATTTCATCCCCATC 25 TCTCAAGGAAGATGGGGATG AGCCCTGGTGATTCTCACAC 26-27 CCAGTGTTAGGGTGTAGCCC GTGTCTGATTCCTAAGCCCG 28-29 TCCAGGGTTGGTCTTAGTGG AGATGTCTTAGCCAGCCCAG 30-31 GTCTTCCATGGCCTTGTTTC CCCTCTGCACCCTAGCTG 32 GTAGATGCTCTGCCCCACTG CTTGGTATCGCAAAGTGCTG

NCAPH 1 AGGCCCGAGGTGGTCTG AGTACTGCTTCGCTAGGGG 2-3 TCAGTGGACAGTGTGTGGTG TAAAACCACCAGCACTAAGATG 4 TGGGTATTCCTATGTAAAGACTTAGC TCATGTATTTGCAGCATTCAAC 5 TGAAGCTTGCTGTGTTTTACC TCAAGTAACTACCTGGTATGCCC 6 CGACAGGTTTTGAAGAACTTGAG TTAGCCCTGCCAAGTCTCAG 7 TCTTTCCCTTGGCTACATGC TGATGGGCTTATTGTACCCC 8 TGAAGGGAGTAAGGAATGCAC GCAGGACCACTCTTGAGCC 9 GTCCCTCCAGTGGTGATTTG AATGCAGTGAGATGCCCG 10 AATGCAGCTCATTTGCGG ACACAACAGGGCTGTGACTG 11 CCTTGATAAGGCTGTCAGCAG GGAACAAGGAAGAATGAAACC 12 AAAGTAAGATGTTTTGCCCACTG GGCACTGCCTAACTGTCATAC 13 AGGAGGAGATTGGGCATAGG AATTGCCAACAGTCTCCCAC 14 GTGTTTCTGAGCTCCTCTGC GCTCAGATTGCGTGACTCTC 15 AGCTCCTTGGTTGAAACTGG AGACATCAACCACTCCCCTC 16 TGAGGCACTGGTTCTCTTTGTAG ACAGGTTGTGTGTGGAAGGC 17 CATACTTGATGAACGAGCTTTTG CTGTATTTAAGAAATCCTATGTTAGCC 18 CACTACCTTGTGACTGTGATTGG GTCCCGGCAACTGAGTAAGG

NCAPH2 1 CGCCTACGCATTTTCCTG AGAGCGCCAGATAGCCATAG 2 AGACCAAAGATGGGTGGGG ACTGTGGTCCCACTGGC 3-4 ACCGATGGATAGCTGGGAAG GGAGCCCAGGCAAACAC 5 TGAGTTTCTTTGGCATGTGG CCCAGGGAAAGTGCTGAC 6-7 ATGTGGGTCCTGTTCTCCAC GTCCAACCAGAAGACCCTCC 8 GGAGAGGCTTGTTGAACACC CTCCCTGGTCCCCTCTTTG 9 GGAAGTAGCCATCAGGGTCC CCAGGGTTGCCAGTTCAC 10 GTACCCAGAGCCTTGAGGG GCAGGCAGCCAGTCCTC 11-12 TACTCCTTTGGGAGGGACG CTGATCCTCCTCCACTGGC 13 GACAGAAGCGCAAGAGGAAG GGGTGACACCAGGTGGG 14-15 CAAAGCCTTGGGTGGACTG AGAGTGAACAGCACGCAGG 16-17 GGCACCTGCCGACTAGC AGGAGCTACTGCCTCCCAAG 18-19 GGGACAGGTGAGCGGTG ACATCAGCAGGGTCTGGG 20 GAGTAGCCTGGGATACGTGG GCCAGCCAGGAGCAGAC

NCAPG 1 GGGCTGGCAGGCTGTAG GGCTGGAGTCACGGTGC 2 TCATGCAAATTGATTGCCC AAAGGAGAACTGGTTTAAAGAGG 3 TGTTGTAACTTCTTGCATTCCG TTTTAAGCTGGGTGGTTGC 4 TGCCCAGCCTATCTTTATCAC AAGAAAATGGCTCCTTGTCC 5 TTGGAGATTCTGGGTAATTTTC CTGGCAGGCTTACTTGACC 6 AAAATACAAGAGATGGAAGCTAGAGG AATATAATCTTAAGCTATGGGGAGG 7 GTGGTTTTAAAGCAAATCTTATTATTC ACTTTGAGGAAGGGGACTGG 8 AATATGACCTGAAAATGGCCC TTTTATACCACTTTAGAGAGCTAGGG 9 GAAAGAACTGAGGCAAATGTAATTAAG GAGACGAACCTAAAACAAAACAGAC 10 TCAATATATGGTGGATACTACTTGGAG TTTCAGAACTGCACAGCAAG 11 CCTGACTCATTAAATTCTGGGC CAAGGAAGCAAATAAAGTCCTACC 12 ACACAAAGCCCTGAGAAAGC GTCCTTTACAGACATCTTATTTAATCC 13 AAAATGACACCTTTGAGGTAATAGAC TCGCCACAGAGTATTTCAGG 14 TTATTGACTGTGAAGATTAACTTTGTC CACACTGGGACCTATCAGGG 15 AGGACAGGCTACATTGAGAGG CATTTTCATCTAGGAAAGAGCC 16 GCTCTTTCCTAGATGAAAATGCAC TGCAGTTCCTCAGTATTTCAAGG 17 GAAGGGGAGCTTATTTTGGG ACCGCATTCCCTAATCTTTG 18 TCAAAGATTAGGGAATGCGG AAACATTTATCAGTAATGCTCTTTCC 19 TGCTAGAAGGACTTTGTCATTTC GGAAACAAAGATAAGAAAACTTGC 20 CATGTGTTGAAAGGATTGTTGTG GCTCTTGATGATGGAATTTTAGAC 21 CACTGATAGAATGTGAGTCAGAAAC TTTGTTCTGATTTTGACAAGGG

NCAPG2 2 TGATTGTGTTTTCCATTAGGG TGGTCGCTAAGGATTCACTG 3 GAATCCCTGTGCTTCTTCCC TTCTTGACTTCTAACTTCAAAATCTG 4 CCCAGGGTGAAGACAATATG AGGCAACAAGAGCGAAACTC 5 GATTTCTCTGAGACTTCCAGTTCC TGTGGCAAGTGGCTACTACG 6 TTGATTTGAAATATGACTTTACTGTTC GTGCAGACAGGAACCATGTG 7 TTTTAGCAATAGCAATGTTTAGCTTAC CCAAATGGAATCTTTCAAAACC 8 TTCACTATGGTTGGAGAGTAGCTG TGGATCTTAAATAACCCTTCTTTG 9 TCAGGTCGTCAGTAGGACACAC CCCCTAACTGATGTCTGTGAAAG 10 CTGTTGCTCTTTCCAAATGC TTTGCTAAATTCAAAAGCACAAG 11 TTTGGGAACACTGTACTTAAGCC AGTACATGCCACAGCCTGG 12 TGAAGCAGATGTGGATTTCG TCTTTGTGGAGTAGGATTACAGG 13 CTACCTGAACCTTTCATTGGC TGTTTGATGAACACATGTCCC 14 CTGTTTCCAGGGTTACAGGC TGTAATTTGTATTATGCTTTTGGG 15 CACCAACATAGGTAGGGAGTGC CTGGGACAAGATGTGACTGG 16 TTCTGAGATGCTGTTACCTTTTAG TTGTTAAATTCTAAACAAACGTAACC 17 GGAATTTACGTAGAGAGACTTTCAG TCAGCACAACAATGGACAGG 18-19 ACCTTTTGACAAAGTTGGGG TGAAAGCTTCACACTTTGATTTG 20-21 ATTGTTCTTGCTGTACGGGG AATCCTGAAACATAACAAACTTGG 22 GTGAGGCTGGATTTACTCCC AGGGAGGGAAGGACTGGTTC 23 GGCTTTTGGTATTGGCTCAC GGCTGATGCATCTGTGTAGG 24 GTGACTCCTTGAGTCCTGGG TTGAGAAACGTAGAGAATGTACTGC 25 TGTTGAAGAAAACCCAAAGC GCTGAGCGCTTACAGCCAC 26 GTGGTTGTGGTTGAACCTTG CAGGAGTAGCCAAGTGCAGG 27 ATAGGGAGGCCCAGCTTAGG CCACCTGGGTGTAAGTAGCAG 28 GGGGATTCTGACTCCATTTTAC CACCTTCCCACATTCCCAC

SMC2 2 TTTGTGGCCTGTTTGATTCC GGGACGTCTCCAAAGTTCAG 3 TGCTGCTTTGTACGGTAGGG ACGTTTTGCAAAGGCATAGG 4 TGACCATGTCATACTTGTTCCAC CAGTGCAAGTAACTAAACCATATTTC 5 TCAGTCTAGATAAACATGAATGGC AATCATTCCCAAATAATGTCTCC 6-7 AAATTCCAGTTGGACACATTG TTTTGGATACATTGCTGACTTTG 8 TGACTTCTGATGTTACGTAAAGTTTC GAAGACAATGCCCAATGAATC 9 CATACAATAAAATGACAGCGTGAAC GCCCAAACAACTGTACTTAGCTTATAG 10 GCTCACTCCAAATTGCTATGG AAACAAACAAGTTCCTTGAGACTG 11 AACCAATCTGAATCTTCGGC AATGTTTCCTACTTGCTTGCG 12 TGCTTTGGAAATAGTGGTTGC AATTTTCACACTGCTTCCAGC 13 CAAAAGAGCTCCTGATGAGTAAAG GCACATGACTTTTCAAGCTAACC 14 CTTGATTGGGATAAATGGCAC GGTTCTGCATATGATTATGTCCC 15 GAAAATTCAGAAAGAGTTTTAATGC GAAGAAGTGGGTAGTTGGGC 16 TCACGTAGGCAAGTCCAGTG CCAGCAACCCTAGCCAAG 17 AAGGTAAGAATCTGAAGTTTGAATG GATACCTAAAGTAAGGTAACGGATATG 18 CCTGTAATTTTCCAAACCATTTC TCAACCAATGATGCTCTACCAC 19 TGGTAACAAAGCTGTGGGTC TGATGACGAGTGAAAAGACTGAG 20 CTGCCTGTAATCACATTGCC ATGCCAGGCTGTAATTTTGC 21 TTAAGAAACTGGCTTATACATATTGG TGCAAAAGGTTAACTCTAATAATTGC 22 TGGCTGCTAAGGAGATAAGGG AAATGGAATTGGGATTTTGTTG 23 CAAAGTAGACCTGCTGTGTTGC AAACACAAATACAGTTTTAAGGTAAGG 24 AGGATCACATATTTGCTTCAGTG ACAATGCTTTAGTATTTATCAACCC 25 TGGACAGCTAGCATTCATTTAGG CCACAATAATTGGACCATAATTAGAC

SMC4 2 GTGGACCGAGATTTCCCC AACTCCGCGCTCAACAAC 3 TGCACTAATGTAGGCTTTTCCC AAATAGGGAGTGAATGAATTTTAGG 4 TGCCTGTGATTAGCAATGAG AAACCAATGCTTTCCGCTG 5 TTTGAAGTAGGCCTATGACTTAATACC TGTAAACCATGATGTGCTGC 6 GGGAAATGAGTGTGTGTAAGTGG CATCCTTTTCCTTTTCCACC 7 GACTTTCATTGTAAATCAGAATGTTTC ATGGGGTCTTGCTATGTTGC 8 GGATTTTCCCCTCATAGCC GGCCACCACTTGATATGCTG 9 TGTTCCAGGTAGGAAATGTGG TCCATGAAAACTAGGTATGATATTCAG 10 AAAAGAAGAACTAGAGGACCCTGAG AGGTTGACTAGTTTTAGGAATGTTG 11 TGGTTTCATTTCAGTGTAGGTTTG AAATGCTGACCTCATTCTTTTG 12 CATACAGGTGCTCAGTTTTGG CTTGAAATATGGTTGTCAAGGC 13 TGAATGAAATGGTCCTGTGC ATGGTGTGAACCCGGGAG 14 CATGAGATGTCTTCCGTTTAGG TTGTACCTGGAAAACAGGGG 15 GGGACAAATCATAGAACAGTCAGG CCTAGTCATTTATAGAAAACGCCATC 16 GGGCGACAGAAGTTTTAGTTTTC CAGTTGAATCCCAATAGTTTCAG 17 TTCTTGAAGATTTTAAGTGCCC GCAATGACTTGCATATACTTAGCTTC 18 CATCACGTCAAGTCATAGCAAC CTTTCCAATGTGAAGGCAGG 19 TCTAAAGTTGAATGATACAAGCAGC ACCAATCTCTCCTCATGCCC 20 TGTAATGTGAAAATGATGGCTC AACAATCCTCCCCAAACCC 21 CACAACTCCTGTTGTTTTCATAGC AACTAAAGATCAGAATTTAGGCTAAGG 22 GGCGTCAGGGCAAGACTAC TGCCTTCACATCCTACCTCC 23 TGTGAAAAGCAAGTTACAGATTCC CATGTGTAATGTTTAGGATACCCC 24 TTCCTGCCATCATTCCTTTAC TGACAACTGCATAAAACAGAAACC

B) RT-PCR primer sequences

Name Forward sequence Reverse sequence

NCAPD3 ex2F, ex4R AGAGACTGGATTGGCTGCAT GGGGCCAGCTCTTCTTTAGA

NCAPD2 ex31F, ex32R CATCCAAACACCCAGCAGC TTCCTAGGATCTGTGCCTGC

C) siRNA oligonucleotide sequences

Name Sequence (sense strand) siLUC CUUACGCUGAGUACUUCGA siNCAPD3 CAUGGAUCUAUGGAGAGUAUU siNCAPH SMARTpool (Dharmacon M-012853-01-0005) siNCAPD2 SMARTpool (Dharmacon M-021198-00-0005)

D) mouse genotyping primer sequences

Name Forward sequence Reverse sequence

NessyGT CAGTGCCCTGGCTACGTACT GTATTCCCCCAAAGAAAACG